Retinal pigment epithelium cell compositions

ABSTRACT

Presented herein are ready to administer (RTA) retinal pigment epithelium (RPE) cell therapy compositions for the treatment of retinal degenerative diseases and injuries. A method of formulating human RPE cells for administration to a subject directly after thawing and of formulating RPE cell therapy compositions for cryopreservation and administration of the cryopreserved composition to a subject subsequent to thawing are also presented. In another aspect, the RTA composition may be formulated as a thaw and inject (TAI) composition, whereby the composition is administered by injection subsequent to thawing.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 62/612,210 filed Dec. 29, 2017, the contents of which ishereby incorporated by reference in its entirety.

BACKGROUND

The retinal pigment epithelium (RPE) is a monolayer ofneuroepithelium-derived pigmented cells that lays on a Bruch's membranebetween the photoreceptor outer segments (POS) and the choroidalvasculature. The RPE monolayer is critical to the function and health ofthe photoreceptors. Dysfunction, injury, and loss of retinal pigmentepithelium (RPE) cells are prominent features of certain eye diseasesand disorders, such as age-related macular degeneration (AMD),hereditary macular degenerations including Best disease (the early onsetform of vitelliform macular dystrophy), and subtypes of retinitispigmentosa (RP). The transplantation of RPE (and photoreceptors) intothe retina of those affected with such diseases can be used as cellreplacement therapy in retinal diseases where RPE have degenerated.

Human fetal and adult RPE have been used as a donor source forallogeneic transplantation. However, practical problems in obtainingsufficient tissue supply and the ethical concerns regarding the use oftissues from aborted fetuses limit widespread use of these donorsources. Given the limitations in the supply of adult and fetal RPEgrafts, the potential of alternative donor sources has been studied.

Human pluripotent stem cells provide significant advantages as a sourceof RPE cells for transplantation. Their pluripotent developmentalpotential enables their so differentiation into authentic functional RPEcells, and given their potential for infinite self-renewal, they canserve as an unlimited donor source of RPE cells. Indeed, it has beendemonstrated that human embryonic stem cells (hESCs) and human inducedpluripotent stem cells (iPSCs) may differentiate into RPE cells invitro, attenuate retinal degeneration and preserve visual function aftersubretinal implantation. Therefore, hESCs can be an unlimited source forthe production of RPE cells for cell therapy.

However, most cell based treatments are usually preserved frozen in acryo-solution that is not compatible with direct administration into thebody, creating a practical problem for clinical use. Cells should betransplanted within hours after they are thawed, or they may begin tolose viability and quality. In addition, cells must be prepared prior toadministration in certified facilities, which may not be in closeproximity to clinical sites, hospitals or other treatment facilities.Finally, each subject's treatment dose must be released by a qualifiedtechnician since preparation of the final formulation is considered tobe part of the cell therapy production process.

The present disclosure addresses these and other shortcomings in thefield of regenerative medicine and RPE cell therapy.

BRIEF SUMMARY

In one aspect, ready to administer (RTA) retinal pigment epithelium(RPE) cell therapy compositions for the treatment of retinaldegenerative diseases and injuries are presented. A method offormulating human RPE cells for administration to a subject directlyafter thawing and of formulating RPE cell therapy compositions forcryopreservation and administration of the cryopreserved composition toa subject subsequent to thawing are also presented. In another aspect,the RTA composition may be formulated as a thaw and inject (TAI)composition, whereby the composition is administered by injectionsubsequent to thawing.

In other aspects, methods for one or more of, slowing the progression ofretinal degenerative disease, slowing the progression of age relatedmacular degeneration (AMD) and/or Geographic Atrophy (GA), preventingretinal degenerative disease, preventing AMD, preventing GA, restoringretinal pigment epithelium (RPE), increasing RPE, replacing RPE ortreating RPE defects, in a subject by administering to the subject acomposition comprising RPE cells and a ready to administer biocompatiblecryopreservation media are presented.

The cryopreservation media described herein may comprise about 2% DMSO,5% DMSO, between about 1% and about 15% DMSO, or between about 0.5% andabout 7% DMSO, or between about 1.5% and about 6.5% DMSO, or betweenabout 1.5% and about 3% DMSO, or between about 4% and about 6% DMSO.

In some aspects, the cryopreservation media comprises: a purinenucleoside (e.g., adenosine), a branched glucan (e.g., dextran-40), azwitterionic organic chemical buffering agent (e.g., HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid))), and a celltolerable polar aprotic solvent (e.g., dimethyl sulfoxide (DMSO).

In other aspects, the retinal degenerative disease comprises one or moreof: RPE dysfunction, photoreceptor dysfunction, accumulation oflipofuscin, formation of drusen, or inflammation.

The retinal degenerative disease may be selected from at least one ofretinitis pigmentosa, lebers congenital amaurosis, hereditary oracquired macular degeneration, age related macular degeneration (AMD),Best disease, retinal detachment, gyrate atrophy, choroideremia, patterndystrophy, RPE dystrophies, Stargardt disease, RPE and retinal damagecaused by any one of photic, laser, infection, radiation, neovascular ortraumatic injury. In addition, the AMD may comprise geographic atrophy(GA).

In another aspect, the RPE defects result from one or more of: advancedage, cigarette smoking, unhealthy body weight, low intake ofantioxidants, or cardiovascular disorders. In yet another aspect, theRPE defects result from a congenital abnormality.

In some aspects, a method of restoring vision in a subject in needthereof is described, including: administering to the subject acomposition comprising: adenosine, dextran-40, lactobionic acid, HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)), sodiumhydroxide, L-glutathione, potassium chloride, potassium bicarbonate,potassium phosphate, dextrose, sucrose, mannitol, calcium chloride,magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), water, and retinal pigment epithelium (RPE) cells.

In other aspects, a method of restoring vision in a subject in needthereof is described, including: administering to the subject acomposition comprising: a cell preservation media comprising: a purinenucleoside (e.g., adenosine), a branched glucan (e.g., dextran-40), azwitterionic organic chemical buffering agent (e.g., HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid))), and a celltolerable polar aprotic solvent (e.g., dimethyl sulfoxide (DMSO); andRPE cells.

In other aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, including: (a) suspending RPE cells to form a cellsuspension in a cell preservation media comprising: a purine nucleoside(e.g., adenosine), a branched glucan (e.g., dextran-40), a zwitterionicorganic chemical buffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); (b) storing the cellsuspension at a cryopreservation temperature; and (c) thawing thecryopreserved suspension, wherein at least about 60% to about 92% of thecells are viable after thawing.

In some aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, including: (a) suspending RPE cells to form a cellsuspension in a cell preservation media comprising: a purine nucleoside(e.g., adenosine), a branched glucan (e.g., dextran-40), a zwitterionicorganic chemical buffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); (b) storing the cellsuspension at a cryopreservation temperature; and (c) thawing thecryopreserved suspension, wherein there is at least about a 50% to abouta 120% yield of cells after thawing.

In some aspects, there was at least about 65% to about 70% yield ofcells after thawing; at least about 64% to about 97% yield of cellsafter thawing; at least about 59% to about 82% yield of cells afterthawing.

In other aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, including: (a) suspending RPE cells to form a cellsuspension in a cell preservation media comprising: a purine nucleoside(e.g., adenosine), a branched glucan (e.g., dextran-40), a zwitterionicorganic chemical buffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); (b) storing the cellsuspension at a cryopreservation temperature; and (c) thawing thecryopreserved suspension, wherein there was at least about a 30% toabout a 112% vitality of cells about twenty-four (24) hours afterthawing.

In some aspects, there was at least about an 89% to about a 110%vitality of cells about twenty-four (24) hours after thawing; there wasat least about a 76% to about a 112% vitality of cells about twenty-four(24) hours after thawing.

In other aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, the method including: (a) suspending RPE cells toform a cell suspension in a cell preservation media comprising: a purinenucleoside (e.g., adenosine), a branched glucan (e.g., dextran-40), azwitterionic organic chemical buffering agent (e.g., HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid))), and a celltolerable polar aprotic solvent (e.g., dimethyl sulfoxide (DMSO); (b)storing the cell suspension at a cryopreservation temperature; and (c)thawing the cryopreserved suspension, wherein there was at least about a3 to about a 7-fold expansion of cells about 8-18 days after thawing andculturing.

In other aspects, there was at least about a 4.2 to about a 5.4-foldexpansion of cells about fourteen (14) days after thawing and culturing.In yet other aspects, there was at least about a 4.2 to about a 4.9-foldexpansion of cells about fourteen (14) days after thawing and culturing;there was at least about a 4.5 to about a 5.4 fold expansion of cellsabout fourteen (14) days after thawing and culturing.

In other aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, the method including: (a) suspending RPE cells toform a cell suspension in a cell preservation media comprising: a purinenucleoside (e.g., adenosine), a branched glucan (e.g., dextran-40), azwitterionic organic chemical buffering agent (e.g., HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid))), and a celltolerable polar aprotic solvent (e.g., dimethyl sulfoxide (DMSO); (b)storing the cell suspension at a cryopreservation temperature; and (c)thawing the cryopreserved cell suspension, wherein the cellsdemonstrated one or more of the following after thawing: a barrierfunction TEER of about 100 Ω to about 1300Ω; a PEDF Upper to Lower Ratioof about 3.5 to about 9.4; a VEGF Lower to Upper Ratio of about 1.2 toabout 5; or a purity of about 95% to about 100%.

In some aspects, the cells had a barrier function of about 107 to about402Ω; or of about 241 to about 715 Ω. In other aspects, the cells had aPEDF upper to lower ratio of about 5.1 to about 9.4; or of about 3.5 toabout 9.4. In other aspects, the cells had a VEGF Lower to Upper Ratioof about 1.2 to about 1.7; or of about 1.2 to about 1.9.

In some aspects, one or more of the purine nucleoside, branched glucan,buffering agent, and the polar aprotic solvent are generally recognizedas safe by the US FDA.

In other aspects, the cell preservation further comprising one or moreof: a sugar acid (e.g., lactobionic acid), one or more of a base (e.g.,sodium hydroxide, potassium hydroxide), an antioxidant (e.g.,L-glutathione), one or more halide salt (e.g., potassium chloride,sodium chloride, magnesium chloride), a basic salt (e.g., potassiumbicarbonate), phosphate salt (e.g., potassium phosphate, sodiumphosphate, potassium phosphate), one or more sugars (e.g., dextrose,sucrose), sugar alcohol, (e.g., mannitol), and water.

In still other aspects, the sugar acid comprises lactobionic acid,glyceric acid, xylonic acid, gluconic acid, ascorbic acid, neuraminicacid, ketodeoxyoctulosonic acid, glucuronic acid, galacturonic acid,galacturonic acid, iduronic acid, tartaric acid, mucic acid, orsaccharic acid.

In some aspects, the one or more of a base comprises sodium hydroxide,or potassium hydroxide. In some aspects, the antioxidant comprisesL-glutathione, ascorbic acid, lipoic acid, uric acid, a carotene,alpha-tocopherol, or ubiquinol. In some aspects, the one or more halidesalt comprises potassium chloride, sodium chloride, or magnesiumchloride. In some aspects, the basic salt comprises potassiumbicarbonate, sodium bicarbonate, or sodium acetate. In some aspects, thephosphate salt comprises potassium phosphate, sodium phosphate, orpotassium phosphate. In some aspects, the one or more sugars comprisesdextrose, sucrose. In some aspects, the sugar alcohol comprisesmannitol, sorbitol, erythritol or xylitol. In some aspects, the one ormore of the sugar acid, base, halide salt, basic salt, antioxidant,phosphate salt, sugars, sugar alcohols are generally recognized as safeby the US FDA.

In other aspects, the RPE composition is administered subretinally. Inother aspects, the RPE composition is administered using a deliverydevice. In other aspects, the delivery device comprises a needle, acapillary and a tip. In other aspects, the delivery device comprises aneedle with an outer diameter of about 0.63 mm and an inner diameter ofabout 0.53 mm, a capillary with an outer diameter of about 0.5 mm and aninner diameter of about 0.25 mm, and a tip with an outer diameter ofabout 0.12 mm and an inner diameter of about 0.07 mm.

In certain aspects, the post-delivery percent viability is between about85% and about 99%, the post-delivery percent recovery is between about65% and about 99%, the post-delivery barrier function TEER is betweenabout 100 and about 600Ω, the PEDF apical/basal ratio is between about 2and about 7, and the post-delivery VEGF basal/apical ratio is betweenabout 1.5 and about 3.

In some aspects, the composition is administered in the subretinalspace. In some aspects, the composition is injected. In some aspects,the composition is administered as a single dose treatment.

In other aspects, the composition does not cause inflammation after itis administered. In yet other aspects, inflammation is characterized bythe presence of cells associated with inflammation. In other aspects,the cell composition is administered without vitrectomy and without theneed to pierce the retina. In some aspects, the cell composition isadministered by a suprachoroidal injection.

In some aspects, the cells secrete one or more of the neurotrophicfactors: fibroblast growth factors (bFGF and aFGF), ciliary neurotrophicfactor (CNTF), pigment epithelium-derived factor (PEDF), brain-derivedneurotrophic factor (BDNF), and vascular endothelial growth factor(VEGF). In some aspects, the cells secrete one or more anti-inflammatorycytokines.

In other aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, including: (a) suspending the RPE cells in a mediacomposition comprising: adenosine, dextran-40, lactobionic acid, HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)), sodiumhydroxide, L-glutathione, potassium chloride, potassium bicarbonate,potassium phosphate, dextrose, sucrose, mannitol, calcium chloride,magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), and water; (b) storing the cell suspension at atemperature adequate for cryopreservation; and (c) thawing thecryopreserved suspension, wherein at least about 60% to about 95% of thecells are viable after thawing.

In other aspects, at least about 40% to about 100% of the cells areviable after thawing; at least about 45% to about 95% of the cells areviable after thawing; at least about 62% to about 70% of the cells areviable after thawing.

In some aspects, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing is described, the method includes: (a) differentiating stemcells into a population of cells comprising RPE cells; (b) enzymaticallyharvesting the RPE cells; (c) neutralizing the enzyme with aneutralizing agent, wherein the neutralizing agent does not comprisehuman serum; (d) suspending the RPE cells in a media compositioncomprising: adenosine, dextran-40, lactobionic acid, HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)), sodiumhydroxide, L-glutathione, potassium chloride, potassium bicarbonate,potassium phosphate, dextrose, sucrose, mannitol, calcium chloride,magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), and water; (e) storing the cell suspension at atemperature adequate for cryopreservation; and (f) thawing thecryopreserved suspension, wherein at least about 70% of the cells areviable after thawing.

In some aspects, the RPE cells are stored in the neutralizing agent forbetween about 1 to about 8 hours and the viability does not decrease bygreater than about 10%. In some aspects, the RPE cells are suspended inthe media composition for about 3 hours prior to cryopreservation, andthe post thaw percent viability does not decrease by greater than about10%, the post thaw percent yield does not decrease by greater than 20%,and the post thaw vitality does not decrease by greater than 10%compared to RPE cells suspended in the media for less than 1 hour.

In some aspects, the RPE cells are suspended in the media compositionfor about 3 hours prior to cryopreservation, and the post thaw barrierfunction does not decrease, the post thaw PEDF upper to lower ratio doesnot decrease by greater than 10%, and the post thaw VEGF lower to upperratio does not decrease compared to RPE cells suspended in the media forless than 1 hour.

In some aspects, the RPE cells are suspended in the media compositionfor between about 2 to 3 hours prior to cryopreservation, and the postthaw percent viability is between about 50 to about 75, the post thawpercent yield is between about 50 to about 95, the post thaw vitality isbetween about 80 to about 120, the post thaw barrier function is about100 to about 750Ω, the post thaw PEDF upper to lower ratio is betweenabout 3 to about 7, and the post thaw VEGF lower to upper ratio isbetween about 1 to 3.

In some aspects, the methods described further comprise: sequentiallyfiltering the RPE cells following step (c), wherein the percentviability is at least 98%. In some aspects, the method furthercomprising: sequentially filtering the RPE cells following step (c) andincubating the RPE cells in the media composition for between about 2-4hours, wherein the percent recovery is between about 80% and about 95%.

In some aspects, the methods described further comprise: sequentiallyfiltering the RPE cells following step (c), incubating the RPE cells inthe neutralizing solution for between about 2 to about 4 hours, andincubating the RPE cells in the media composition for between about 2-4hours, wherein the percent viability is between about 80% and about 99%and wherein the percent recovery is between about 70% and about 95%.

In other aspects, the methods described further comprise: sequentiallyfiltering the RPE cells following step (c), incubating the RPE cells inthe neutralizing solution for between about 2 to about 4 hours, andincubating the RPE cells in the media composition for between about 2-4hours, wherein the post thaw percent viability is between about 80% andabout 99%, the post thaw percent recovery is between about 70% and about95% and the PEDF secretion is between about 2,000 ng/ml/day and about3,000 ng/ml/day.

In other aspects, the methods described further comprise: incubating theRPE cells in the media composition for between about 2-6 hours at roomtemperature, wherein the percent viability is between about 80% andabout 99% and wherein the percent recovery is between about 80% andabout 120%.

In other aspects, a composition is described comprising: (a) adenosine,dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater; and (b) RPE cells, wherein the composition can be stored atcryothermic temperatures and wherein the composition is ready toadminister to a subject directly after thawing.

In other aspects, a composition is described including: (a) a cellpreservation media comprising: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); and (b) RPE cells.

In certain compositions, the one or more of the purine nucleoside,branched glucan, buffering agent, and the polar aprotic solvent aregenerally recognized as safe by the US FDA.

In other aspects, the therapeutic cell compositions described hereinfurther comprise: one or more of: a sugar acid (e.g., lactobionic acid),one or more of a base (e.g., sodium hydroxide, potassium hydroxide), anantioxidant (e.g., L-glutathione), one or more halide salt (e.g.,potassium chloride, sodium chloride, magnesium chloride), a basic salt(e.g., potassium bicarbonate), phosphate salt (e.g., potassiumphosphate, sodium phosphate, potassium phosphate), one or more sugars(e.g., dextrose, sucrose), sugar alcohol, (e.g., mannitol), and water.

In other aspects of the compositions described, the sugar acid compriseslactobionic acid, glyceric acid, xylonic acid, gluconic acid, ascorbicacid, neuraminic acid, ketodeoxyoctulosonic acid, glucuronic acid,galacturonic acid, galacturonic acid, iduronic acid, tartaric acid,mucic acid, or saccharic acid. In other aspects of the compositionsdescribed, the one or more of a base comprises sodium hydroxide, orpotassium hydroxide. In other aspects of the compositions described, theantioxidant comprises L-glutathione, ascorbic acid, lipoic acid, uricacid, a carotene, alpha-tocopherol, or ubiquinol. In other aspects ofthe compositions described, the one or more halide salt comprisespotassium chloride, sodium chloride, or magnesium chloride. In otheraspects of the compositions described, the basic salt comprisespotassium bicarbonate, sodium bicarbonate, or sodium acetate. In otheraspects of the compositions described, the phosphate salt comprisespotassium phosphate, sodium phosphate, or potassium phosphate. In otheraspects of the compositions described, the one or more sugars comprisesdextrose, sucrose. In other aspects of the compositions described, thesugar alcohol comprises mannitol, sorbitol, erythritol or xylitol. Inother aspects of the compositions described, the one or more of thesugar acid, base, halide salt, basic salt, antioxidant, phosphate salt,sugars, sugar alcohols are generally recognized as safe by the US FDA.

In other aspects of the compositions described, the RPE cellconcentration is between about 100,000 and about 10,000,000 cells/ml. Inother aspects of the compositions described, the number of cells in saidcomposition is between about 100,000 to about 500,000.

In other aspects of the compositions described, the cell preservationmedia further comprises one or more of: a sugar acid (e.g., lactobionicacid), one or more of a base (e.g., sodium hydroxide, potassiumhydroxide), an antioxidant (e.g., L-glutathione), one or more halidesalt (e.g., potassium chloride, sodium chloride, magnesium chloride), abasic salt (e.g., potassium bicarbonate), phosphate salt (e.g.,potassium phosphate, sodium phosphate, potassium phosphate), one or moresugars (e.g., dextrose, sucrose), sugar alcohol, (e.g., mannitol), andwater.

In other aspects of the compositions described, the compositions furthercomprise one or more of ROCK inhibitor or NA.

In further aspects, the cryopreservation media comprises: adenosine,dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater.

In some embodiments, the cryopreservation media includes about 2% DMSO.In other embodiments, the cryopreservation media includes about 5% DMSO.In yet other embodiments, the cryopreservation media includes betweenabout 1% and about 15% DMSO.

In further embodiments, the cryopreservation media includes: a purinenucleoside (e.g., adenosine), a branched glucan (e.g., dextran-40), azwitterionic organic chemical buffering agent (e.g., HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid))), and a celltolerable polar aprotic solvent (e.g., dimethyl sulfoxide (DMSO). Instill further embodiments, one or more of the purine nucleoside,branched glucan, buffering agent, and the polar aprotic solvent aregenerally recognized as safe by the US FDA.

In some embodiments, the cryopreservation media further includes one ormore of: a sugar acid (e.g., lactobionic acid), one or more of a base(e.g., sodium hydroxide, potassium hydroxide), an antioxidant (e.g.,L-glutathione), one or more halide salt (e.g., potassium chloride,sodium chloride, magnesium chloride), a basic salt (e.g., potassiumbicarbonate), phosphate salt (e.g., potassium phosphate, sodiumphosphate, potassium phosphate), one or more sugars (e.g., dextrose,sucrose), sugar alcohol, (e.g., mannitol), and water.

In other embodiments, one or more of the sugar acid, base, halide salt,basic salt, antioxidant, phosphate salt, sugars, sugar alcohols aregenerally recognized as safe by the US FDA.

In certain embodiments, the retinal degenerative disease may be one ormore of: RPE dysfunction, photoreceptor dysfunction, accumulation oflipofuscin, formation of drusen, or inflammation.

In other embodiments, the retinal degenerative disease is selected fromat least one of retinitis pigmentosa, lebers congenital amaurosis,hereditary or acquired macular degeneration, age related maculardegeneration (AMD), Best disease, retinal detachment, gyrate atrophy,choroideremia, pattern dystrophy, RPE dystrophies, Stargardt disease,RPE and retinal damage caused by any one of photic, laser, infection,radiation, neovascular or traumatic injury. In yet other embodiments,the AMD is geographic atrophy (GA).

In certain embodiments, the RPE defects may result from one or more of:advanced age, cigarette smoking, unhealthy body weight, low intake ofantioxidants, or cardiovascular disorders. In other embodiments, the RPEdefects may result from a congenital abnormality.

In other embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells in a composition comprising:adenosine, dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater, storing the cell suspension at a temperature adequate forcryopreservation and thawing the cryopreserved suspension, wherein atleast about 70% of the cells are viable after thawing.

In other embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells to form a cell suspension ina media which includes: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); storing the cellsuspension at a cryopreservation temperature; and thawing thecryopreserved suspension, wherein at least about 60% to about 75% of thecells are viable after thawing.

In other embodiments a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes the addition of one or more of: a sugar acid (e.g.,lactobionic acid), one or more of a base (e.g., sodium hydroxide,potassium hydroxide), an antioxidant (e.g., L-glutathione), one or morehalide salt (e.g., potassium chloride, sodium chloride, magnesiumchloride), a basic salt (e.g., potassium bicarbonate), phosphate salt(e.g., potassium phosphate, sodium phosphate, potassium phosphate), oneor more sugars (e.g., dextrose, sucrose), sugar alcohol, (e.g.,mannitol), and water to the formulation.

In some embodiments, at least about 40% to about 100% of the cells areviable after thawing; at least about 45% to about 95% of the cells areviable after thawing; at least about 62% to about 70% of the cells areviable after thawing.

In some embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells to form a cell suspension ina media, which includes: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); storing the cellsuspension at a cryopreservation temperature; and thawing thecryopreserved suspension, wherein there was at least about a 59% toabout a 92% yield of cells after thawing.

In some embodiments, there was at least about 65% to about 70% yield ofcells after thawing; at least about 64% to about 92% yield of cellsafter thawing; at least about 59% to about 82% yield of cells afterthawing.

In some embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells to form a cell suspension ina media, which includes: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); storing the cellsuspension at a cryopreservation temperature; and thawing thecryopreserved suspension, wherein there was at least about a 76% toabout a 112% vitality of cells about twenty-four (24) hours afterthawing.

In some embodiments, there was at least about an 89% to about a 110%vitality of cells about twenty-four (24) hours after thawing; there wasat least about a 76% to about a 112% vitality of cells about twenty-four(24) hours after thawing.

In other embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells to form a cell suspension ina media, which includes: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); storing the cellsuspension at a cryopreservation temperature; and thawing thecryopreserved suspension, wherein there was at least about a 4.2 toabout a 5.4 fold expansion of cells about fourteen (14) days afterthawing and culturing.

In some embodiments, there was at least about a 4.2 to about a 4.9 foldexpansion of cells about fourteen (14) days after thawing and culturing;there was at least about a 4.5 to about a 5.4 fold expansion of cellsabout fourteen (14) days after thawing and culturing. In someembodiments, there was at least about a 3 to about a 7 fold expansion ofcells about 8-18 days after thawing and culturing. In some embodiments,there was at least about a 3 to about a 5 fold expansion of cells about8 days after thawing and culturing.

In some embodiments, a method of formulating human retinal pigmentepithelium (RPE) cells for administration to a subject directly afterthawing includes: suspending the RPE cells to form a cell suspension ina media, which includes: a purine nucleoside (e.g., adenosine), abranched glucan (e.g., dextran-40), a zwitterionic organic chemicalbuffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); storing the cellsuspension at a cryopreservation temperature; and thawing thecryopreserved suspension, wherein the cells demonstrated one or more ofthe following after thawing: had a barrier function of about 100 toabout 720; had a PEDF Upper to Lower Ratio of about 3.5 to about 9.4;had a VEGF Lower to Upper Ratio of about 1.2 to about 2.7; had a purityof about 95 to about 100%; had a potency of about 150 to about 900.

In some embodiments, the cells had a barrier function of about 107 toabout 402Ω; or of about 241 to about 715Ω.

In some embodiments, the cells had a PEDF Upper to Lower Ratio of about5.1 to about 9.4; or of about 3.5 to about 9.4.

In some embodiments, the cells had a VEGF Lower to Upper Ratio of about1.2 to about 1.7; or of about 1.2 to about 1.9.

In some embodiments, a method of restoring vision in a subject in needthereof, includes: administering to the subject a composition including:adenosine, dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), water,and retinal pigment epithelium cells.

In some embodiments, a method of restoring vision in a subject in needthereof, includes: administering to the subject a composition including:a cell preservation media comprising: a purine nucleoside (e.g.,adenosine), a branched glucan (e.g., dextran-40), a zwitterionic organicchemical buffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); and RPE cells. In someembodiments, the cell preservation media further comprises one or moreof: a sugar acid (e.g., lactobionic acid), one or more of a base (e.g.,sodium hydroxide, potassium hydroxide), an antioxidant (e.g.,L-glutathione), one or more halide salt (e.g., potassium chloride,sodium chloride, magnesium chloride), a basic salt (e.g., potassiumbicarbonate), phosphate salt (e.g., potassium phosphate, sodiumphosphate, potassium phosphate), one or more sugars (e.g., dextrose,sucrose), sugar alcohol, (e.g., mannitol), and water.

In some embodiments, the cell composition is administered in thesubretinal space. In other embodiments, the cell composition isinjected. In some embodiments, the cell composition may be administeredinto the subretinal space transvitreally.

In some embodiments, the cell composition is administered as a singledose treatment. In some embodiments, the single dose treatment comprisesa single administration comprising several injections. In someembodiments, the injections comprise the administration of severalsubretinal blebs.

In some embodiments, the cell composition is administered to thesubretinal space without vitrectomy and without the need to pierce theretina. In some embodiments, the cell composition is administered bysuprachoroidal injection.

In some embodiments RPE cells secrete a variety of neurotrophic factors,such as fibroblast growth factors (bFGF and aFGF), ciliary neurotrophicfactor (CNTF), pigment epithelium-derived factor (PEDF), brain-derivedneurotrophic factor (BDNF), vascular endothelial growth factor (VEGF)and others, that help to maintain the structural integrity ofchoriocapillaris endothelium and photoreceptors. RPE cells also secreteanti-inflammatory cytokines such as transforming growth factor (TGF)-β,important in establishing the immune privileged properties of the eye.The RPE cells used in the RTA therapeutic cell compositions describedherein are capable of secreting neurotrophic factors.

In some embodiments, the cell composition does not cause inflammationafter it is administered. In some embodiments, a mild inflammation maybe characterized by the presence of cells associated with inflammation.

In some embodiments, a composition includes: (a) adenosine, dextran-40,lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater; and (b) RPE cells, wherein the composition can be stored atcryothermic temperatures and wherein the composition is ready toadminister to a subject directly after thawing.

In other embodiments, a therapeutic cell composition may comprise: acell preservation media including: a purine nucleoside (e.g.,adenosine), a branched glucan (e.g., dextran-40), a zwitterionic organicchemical buffering agent (e.g., HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))), and a cell tolerable polaraprotic solvent (e.g., dimethyl sulfoxide (DMSO); and RPE cells.

In some embodiments, the cell preservation media further comprises oneor more of: a sugar acid (e.g., lactobionic acid), one or more of a base(e.g., sodium hydroxide, potassium hydroxide), an antioxidant (e.g.,L-glutathione), one or more halide salt (e.g., potassium chloride,sodium chloride, magnesium chloride), a basic salt (e.g., potassiumbicarbonate), phosphate salt (e.g., potassium phosphate, sodiumphosphate, potassium phosphate), one or more sugars (e.g., dextrose,sucrose), sugar alcohol, (e.g., mannitol), and water.

In yet other embodiments, the cell preservation media may comprise oneor more of ROCK inhibitor and NA.

In some embodiments, an RPE cell composition comprises adenosine,dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater; and RPE cells at a cell concentration of between about 2,000,000and about 5,000,000 cells/ml. The composition can be stored atcryothermic temperatures, and the composition is ready to administer toa subject directly after thawing. In this RPE cell composition thenumber of cells may be between about 200,000 to about 500,000. Inaddition, the volume administered to the subject may be between about 50μl and about 100 μl.

Further aspects of the technology described herein will be brought outin the following portions of the specification, wherein the detaileddescription is for the purpose of fully disclosing preferred embodimentsof the technology without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The technology described herein will be more fully understood byreference to the following drawings which are for illustrative purposesonly:

FIG. 1 is a graph showing the viability and vitality of retinal pigmentepithelium (RPE) cells after thawing. Cells were cryopreserved incryopreservation media with 5% DMSO (CS5) prior to thawing.

FIG. 2 is a histological image of the eye of a naive animal (untreatedanimal) showing no pathology (H&E stained at ×4 magnification field).

FIG. 3 is a histological image of the right eye (non-treated/controleye) of an animal in the control group showing no pathological changes.(H&E stained at ×4 magnification field).

FIG. 4A is a histological image of the left eye (treated eye) taken froman animal treated with BSS Plus and sacrificed on day 1 of the study,showing mild inflammation with mild infiltration of the sclera. (H&Estained at ×4 magnification field).

FIG. 4B is a histological image of the left eye (treated eye) taken froman animal treated with BSS Plus and sacrificed on day 1 of the study,showing mild inflammation and a few lose macrophages and lymphocytes.(H&E stained at ×20 magnification field).

FIG. 5A is a histological image of the left eye (treated eye) taken froman animal treated with CS5 and sacrificed on day 1 of the study, showingmoderate inflammation and infiltration of the sclera. (H&E stained at ×4magnification field).

FIG. 5B is a histological image of the left eye (treated eye) taken froman animal treated with CS5 and sacrificed on day 1 of the study, showingmoderate inflammation with some macrophages and few neutrophils. (H&Estained at ×20 magnification field).

FIG. 6A is a histological image of the left eye (treated eye) taken froman animal treated with CS2 and sacrificed on day 1 of the study, showingmoderate inflammation with macrophages and neutrophils in the cornea.(H&E stained at ×4 magnification field).

FIG. 6B is a histological image of the left eye (treated eye) taken froman animal treated with CS2 and sacrificed on day 1 of the study, showingmoderate inflammation with macrophages and neutrophils. (H&E stained at×20 magnification field).

FIG. 7A is a histological image of the left eye (treated eye) taken froman animal treated with BSS PLUS:CS2 and sacrificed on day 1 of thestudy, showing strong inflammation with moderate infiltration of thesclera. (H&E stained at ×4 magnification field).

FIG. 7B is a histological image of the left eye (treated eye) taken froman animal treated with BSS PLUS:CS2 and sacrificed on day 1 of thestudy, showing strong inflammation with fibrin shown at the lower rightcorner next to the lymphocytes in the sclera. (H&E stained at ×20magnification field).

FIG. 8A is a histological image of the left eye (treated eye) taken froman animal treated with BSS PLUS and sacrificed on day 3 of the study,showing moderate inflammation with moderate infiltration of the sclera.(H&E stained at ×4 magnification field).

FIG. 8B is a histological image of the left eye (treated eye) taken froman animal treated with BSS PLUS and sacrificed on day 3 of the study,showing moderate inflammation with several macrophages. (H&E stained at×20 magnification field).

FIG. 9A is a histological image of the left eye (treated eye) taken froman animal treated with CS5 and sacrificed on day 3 of the study, showingstrong inflammation with a focal granulation reaction. (H&E stained at×4 magnification field).

FIG. 9B is a histological image of the left eye (treated eye) taken froman animal treated with CS5 and sacrificed on day 3 of the study, showingstrong inflammation with several macrophages and fibroblasts,demonstrating an early stage, transitory foreign body reaction. (H&Estained at ×20 magnification field).

FIG. 10A is a histological image of the left eye (treated eye) takenfrom an animal treated with CS2 and sacrificed on day 3 of the study,showing strong inflammation with a focal granulation reaction. (H&Estained at ×4 magnification field).

FIG. 10B is a histological image of the left eye (treated eye) takenfrom an animal treated with CS2 and sacrificed on day 3 of the study,showing strong inflammation with several macrophages and fibroblasts,demonstrating an early stage, transitory foreign body reaction. (H&Estained at ×20 magnification field).

FIG. 11A is a histological image of the left eye (treated eye) takenfrom an animal treated with BSS PLUS:CS2 and sacrificed on day 3 of thestudy, showing mild inflammation with mild edema. (H&E stained at ×4magnification field).

FIG. 11B is a histological image of the left eye (treated eye) takenfrom an animal treated with BSS PLUS:CS2 and sacrificed on day 3 of thestudy, showing mild inflammation and few macrophages. (H&E stained at×20 magnification field).

FIG. 12A is a histological image of the left eye (treated eye) takenfrom an animal treated with BSS PLUS and sacrificed on day 10 of thestudy, showing mild inflammation with few macrophages. (H&E stained at×4 magnification field).

FIG. 12B is a histological image of the left eye (treated eye) takenfrom an animal treated with BSS PLUS and sacrificed on day 10 of thestudy, showing mild inflammation with few macrophages. (H&E stained at×20 magnification field).

FIG. 13A is a histological image of the left eye (treated eye) takenfrom an animal treated with CS5 and sacrificed on day 10 of the study,showing mild inflammation with few macrophages. (H&E stained at ×4magnification field).

FIG. 13B is a histological image of the left eye (treated eye) takenfrom an animal treated with CS5 and sacrificed on day 10 of the study,showing mild inflammation with few macrophages. (H&E stained at ×20magnification field).

FIG. 14A is a histological image of the left eye (treated eye) takenfrom an animal treated with CS2 and sacrificed on day 10 of the study,showing mild inflammation with few macrophages. (H&E stained at ×4magnification field).

FIG. 14B is a histological image of the left eye (treated eye) takenfrom an animal treated with CS2 and sacrificed on day 10 of the study,showing mild inflammation with few macrophages. (H&E stained at ×20magnification field).

FIG. 15 is an illustration of the RPE and adjacent cells.

FIG. 16 is a graph of the viability of Group 2 (G2) (NUTS(−)+HSA) andGroup 3 (G3) (NUTS(−)) at 4° C. over time post-filtration compared tothe control group, G1. Filtered cell compositions were sampled andcounted (n=3) at three-time points: 0 hours post-filtration, after 2hours and after 4 hours.

FIG. 17A is a graph showing the effect of 0 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell viability, prior to cryopreservation.

FIG. 17B is a graph showing the effect of 0 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell recovery, prior to cryopreservation.

FIG. 18A is a graph showing the effect of 2 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell viability, prior to cryopreservation.

FIG. 18B is a graph showing the effect of 2 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell recovery, prior to cryopreservation.

FIG. 19A is a graph showing the effect of 4 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell viability, prior to cryopreservation.

FIG. 19B is a graph showing the effect of 4 hours incubation of thetherapeutic cell compositions post-filtration followed by 0, 2, 3, and 4hours incubation of the therapeutic cell compositions incryopreservation medium on cell recovery, prior to cryopreservation.

FIG. 20A is a graph showing the effect of prolonged pre-cryopreservationincubation of cell compositions in cryomedium for 0, 2, 3, and 4 on cellviability post-thawing.

FIG. 20B is a graph showing the effect of prolonged pre-cryopreservationincubation of cell compositions in cryomedium for 0, 2, 3, and 4 on cellrecovery post-thawing.

FIG. 21A is a graph showing the effect of therapeutic cell compositionsincubated in enzyme neutralizing solution pre-cryopreservation followedby incubation in cryomedium pre-cryopreservation. Cells were thencryopreserved, thawed and analyzed for post-thaw viability.

FIG. 21B is a graph showing the effect of therapeutic cell compositionsincubated in enzyme neutralizing solution pre-cryopreservation followedby incubation in cryomedium pre-cryopreservation. Cells were thencryopreserved, thawed and analyzed for post-thaw recovery.

FIG. 22 is a graph showing the percent recovery of therapeutic cellspost-filtration and incubation for 2 hours at about 2-8° C. and at RT.

FIG. 23A is a graph showing the viability of therapeutic cellcompositions comprising cryomedium under different incubationconditions.

FIG. 23B is a graph showing the viability of therapeutic cellcompositions comprising cryomedium under different incubationconditions.

FIG. 24A is a graph showing viability of thawed RTA cell compositionskept at room temperature over a 4-hour period. Cells were tested at timepoints 0, 2, 4 hrs.

FIG. 24B is a graph showing recovery of thawed RTA cell compositionskept at room temperature over a 4-hour time period. Cells were tested attime points 0, 2, 4 hrs.

FIG. 25A is a histological image of the treated eye taken from an animaltreated with RTA (cells+CS5) and sacrificed on day 14 of the study,showing mild inflammation and a few lose macrophages and lymphocytes.(H&E stained at ×4 magnification field).

FIG. 25B is a histological image of the treated eye taken from an animaltreated with RTA (cells+CS5) and sacrificed on day 14 of the study,showing mild inflammation and a few lose macrophages and lymphocytes.(H&E stained at ×20 magnification field).

DETAILED DESCRIPTION

Compositions described herein may be used as a ready to administer (RTA)retinal pigment epithelium (RPE) cell composition suitable fortherapeutic use which does not require preparation procedures such aswashing or reconstitution prior to injection or implantation into asubject's eye. In some embodiments, the cell therapy composition ispreserved in a non-toxic cryo-solution, shipped to the clinical site,thawed and readily administered to the subject's eye by healthcarepersonnel. By eliminating preparation procedures prior toadministration, especially those preparation procedures that must becarried out under GLP/GMP conditions, widespread access to RPE celltherapy can be made available, while preserving product safety andquality.

“Retinal pigment epithelium cells”, “RPE cells”, “RPEs”, which may beused interchangeably as the context allows, refers to cells of a celltype that is for example, functionally, epi-genetically, or byexpression profile similar to that of native RPE cells which form thepigment epithelium cell layer of the retina (e.g., upon transplantation,administration or delivery within an eye, they exhibit functionalactivities similar to those of native RPE cells).

According to some embodiments, the RPE cell expresses at least one, two,three, four or five markers of mature RPE cells. According to someembodiments, the RPE cell expresses between at least two to at least tenor at least two to at least thirty markers of mature RPE cells. Suchmarkers include, but are not limited to CRALBP, RPE65, PEDF, PMEL17,bestrophin 1 and tyrosinase. Optionally, the RPE cell may also express amarker of a RPE progenitor (e.g., MITF). In other embodiments, the RPEcells express PAX-6. In other embodiments, the RPE cells express atleast one marker of a retinal progenitor cell including, but not limitedto Rx, OTX2 or SIX3. Optionally, the RPE cells may express either SIX6and/or LHX2.

As used herein the phrase “markers of mature RPE cells” refers toantigens (e.g., proteins) that are elevated (e.g., at least 2-fold, atleast 5-fold, at least 10-fold) in mature RPE cells with respect to nonRPE cells or immature RPE cells.

As used herein the phrase “markers of RPE progenitor cells” refers toantigens (e.g., proteins) that are elevated (e.g., at least 2-fold, atleast 5-fold, at least 10-fold) in RPE progenitor cells when comparedwith non RPE cells.

According to other embodiments, the RPE cells have a morphology similarto that of native RPE cells which form the pigment epithelium cell layerof the retina. For example, the cells may be pigmented and have acharacteristic polygonal shape.

According to still other embodiments, the RPE cells are capable oftreating diseases such as macular degeneration.

According to additional embodiments, the RPE cells fulfill at least 1,2, 3, 4 or all of the requirements listed herein above.

As used herein, the phrase “stem cells” refers to cells which arecapable of remaining in an undifferentiated state (e.g., pluripotent ormultipotent stem cells) for extended periods of time in culture untilinduced to differentiate into other cell types having a particular,specialized function (e.g., fully differentiated cells). Preferably, thephrase “stem cells” encompasses embryonic stem cells (ESCs), inducedpluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cellsand hematopoietic stem cells.

According to some embodiments, the RPE cells are generated frompluripotent stem cells (e.g., ESCs or iPSCs).

Induced pluripotent stem cells (iPSCs) can be generated from somaticcells by genetic manipulation of somatic cells, e.g., by retroviraltransduction of somatic cells such as fibroblasts, hepatocytes, gastricepithelial cells with transcription factors such as Oct-3/4, Sox2,c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, 1(1):39-49; Aoi T, etal., Generation of Pluripotent Stem Cells from Adult Mouse Liver andStomach Cells. Science. 2008 Feb. 14. (Epub ahead of print); IH Park,Zhao R, West J A, et al. Reprogramming of human somatic cells topluripotency with defined factors. Nature 2008; 451:141-146; KTakahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stemcells from adult human fibroblasts by defined factors. Cell 2007;131:861-872]. Other embryonic-like stem cells can be generated bynuclear transfer to oocytes, fusion with embryonic stem cells or nucleartransfer into zygotes if the recipient cells are arrested in mitosis. Inaddition, iPSCs may be generated using non-integrating methods e.g., byusing small molecules or RNA.

The phrase “embryonic stem cells” refers to embryonic cells that arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apreimplantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO 2006/040763) and embryonic germ (EG) cells which are obtained fromthe genital tissue of a fetus any time during gestation, preferablybefore 10 weeks of gestation. The embryonic stem cells of someembodiments of the present disclosure can be obtained using well-knowncell-culture methods. For example, human embryonic stem cells can beisolated from human blastocysts.

Human blastocysts are typically obtained from human in vivopreimplantation embryos or from in vitro fertilized (IVF) embryos.Alternatively, a single cell human embryo can be expanded to theblastocyst stage. For the isolation of human ES cells the zona pellucidais removed from the blastocyst and the inner cell mass (ICM) is isolatedby a procedure in which the trophectoderm cells are lysed and removedfrom the intact ICM by gentle pipetting. The ICM is then plated in atissue culture flask containing the appropriate medium which enables itsoutgrowth. Following 9 to 15 days, the ICM derived outgrowth isdissociated into clumps either by a mechanical dissociation or by anenzymatic degradation and the cells are then re-plated on a fresh tissueculture medium. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and re-plated. Resulting ES cells are then routinely split every4-7 days. For further details on methods of preparation human ES cells,see Reubinoff et al. Nat Biotechnol 2000, May: 18(5): 559; Thomson etal., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev.Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongsoet al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril.69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used according to some embodiments of the present disclosure Human EScells can be purchased from the NIH human embryonic stem cells registry,www.grants.nih.govstem_cells/ or from other hESC registries.Non-limiting examples of commercially available embryonic stem celllines are HAD-C I02, ESI, BGO I, BG02, BG03, BG04, CY12, CY30, CY92,CYIO, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11,CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8,HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16,HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24,HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WAO I, UCSF4, NYUES I,NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3,WA077 (H7), WA09 (H9), WA I3 (HI3), WA14 (HI4), HUES 62, HUES 63, HUES64, CT I, CT2, CT3, CT4, MA135, Eneavour-2, WIBR I, WIBR2, WIBR3, WIBR4,WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNhemI9, BJNhem20, SAOO I,SAOOI.

According to some embodiments, the embryonic stem cell line is HAD-C102or ESI.

In addition, ES cells can be obtained from other species, includingmouse (Mills and Bradley, 2001), golden hamster [Doetschman et al.,1988, Dev Biol. 127: 224-7], rat [Lannaccone et al., 1994, Dev Biol.163: 288-92], rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 30 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine days (and preferably not longer thanfourteen days) post fertilization (i.e., prior to the gastrulationevent) in vitro using standard embryonic stem cell culturing methods.

Another method for preparing ES cells is described in Chung et al., CellStem Cell, Volume 2, Issue 2, 113-117, 7 Feb. 2008. This methodcomprises removing a single cell from an embryo during an in vitrofertilization process. The embryo is not destroyed in this process.

EG (embryonic germ) cells can be prepared from the primordial germ cellsobtained from fetuses of about 8-11 weeks of gestation (in the case of ahuman fetus) using laboratory techniques known to anyone skilled in thearts. The genital ridges are dissociated and cut into small portionswhich are thereafter disaggregated into cells by mechanicaldissociation. The EG cells are then grown in tissue culture flasks withthe appropriate medium. The cells are cultured with daily replacement ofmedium until a cell morphology consistent with EG cells is observed,typically after 7-30 days or 1-4 passages. For additional details onmethods of preparing human EG cells, see Shamblott et al., [Proc. Natl.Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622 incorporatedherein by reference in their entirety.

Yet another method for preparing ES cells is by parthenogenesis. Theembryo is also not destroyed in the process.

ES culturing methods may include the use of feeder cell layers whichsecrete factors needed for stem cell proliferation, while at the sametime, inhibiting their differentiation. The culturing is typicallycarried out on a solid surface, for example a surface coated withgelatin or vimentin. Exemplary feeder layers include human embryonicfibroblasts, adult fallopian epithelial cells, primary mouse embryonicfibroblasts (PMEF), mouse embryonic fibroblasts (MEF), murine fetalfibroblasts (MFF), human embryonic fibroblast (HEF), human fibroblastsobtained from the differentiation of human embryonic stem cells, humanfetal muscle cells (HFM), human fetal skin cells (HFS), human adult skincells, human foreskin fibroblasts (HFF), human umbilical cordfibroblasts, human cells obtained from the umbilical cord or placenta,and human marrow stromal cells (hMSCs). Growth factors may be added tothe medium to maintain the ESCs in an undifferentiated state. Suchgrowth factors include bFGF and/or TGF. In another embodiment, agentsmay be added to the medium to maintain the hESCs in a naiveundifferentiated state; see for example Kalkan et al., 2014, Phil.Trans. R. Soc. B, 369: 20130540.

Human umbilical cord fibroblasts may be expanded in Dulbecco's ModifiedEagle's Medium (e.g. DMEM, SH30081.01, Hyclone) supplemented with humanserum (e.g. 20%) and glutamine. Preferably the human cord cells areirradiated. This may be effected using methods known in the art (e.g.Gamma cell, 220 Exel, MDS Nordion 3,500-7500 rads). Once sufficientcells are obtained, they may be frozen (e.g. cryopreserved). Forexpansion of ESCs, the human cord fibroblasts may be seeded on a solidsurface (e.g. T75 or T I75 flasks) optionally coated with an adherentsubstrate such as gelatin (e.g. recombinant human gelatin (RhG 100-001,Fibrogen) or human Vitronectin or Laminin 521 (Bio lamina) at aconcentration of about 25,000-100,000 cells/cm² in DMEM (e.g.SH30081.01, Hyclone) supplemented with about 20% human serum (andglutamine). hESCs can be plated on top of the feeder cells 1-4 dayslater in a supportive medium (e.g. NUTRISTEM® or NUT(+) with human serumalbumin). Additional factors may be added to the medium to preventdifferentiation of the ESCs such as bFGF and TGFβ. Once a sufficientamount of hESCs are obtained, the cells may be mechanically disrupted(e.g. by using a sterile tip or a disposable sterile stem cell tool;14602 Swemed). For example, the cells may be expanded mechanicallyduring weekly passaging. Alternatively, the cells may be removed byenzymatic treatment (e.g. collagenase A, or TrypLE Select). This processmay be repeated several times to reach the necessary concentration ofhESC. According to some embodiments, following the first round ofexpansion, the hESCs are removed using TrypLE Select and following thesecond round of expansion, the hESCs are removed using collagenase A.

The ESCs may be expanded on feeders prior to the differentiation step.Exemplary feeder layer based cultures are described herein above. Theexpansion is typically carried out for at least two days, three days,four days, five days, six days, seven days, eight days, nine days, orten days. The expansion can be carried out for at least 1 passage, atleast 2 passages, at least 3 passages, at least 4 passages, at least 5passages, at least 6 passages, at least 7 passages, at least 8 passages,at least 9 passages or at least 10 passages. In some embodiments, theexpansion is carried out for at least 2 passages to at least 20passages. In other embodiments, the expansion is performed for at least2 to at least 40 passages. Following expansion, the pluripotent stemcells (e.g. ESCs) may be subjected to directed differentiation using adifferentiating agent.

Feeder cell free systems can also be used in ES cell culturing. Suchsystems utilize matrices supplemented with serum replacement, cytokinesand growth factors (including IL6 and soluble IL6 receptor chimera) as areplacement for the feeder cell layer. Stem cells can be grown on asolid surface such as an extracellular matrix (e.g., MATRIGELR™, lamininor vitronectin) in the presence of a culture medium —for example theLonza L7 system, mTeSR, StemPro, XFKSR, E8, NUTRISTEM®). Unlikefeeder-based cultures which require the simultaneous growth of feedercells and stem cells and which may result in mixed cell populations,stem cells grown on feeder-free systems are easily separated from thesurface. The culture medium used for growing the stem cells containsfactors that effectively inhibit differentiation and promote theirgrowth such as MEF-conditioned medium and bFGF.

In some embodiments, following expansion, the pluripotent ESCs aresubjected to directed differentiation on an adherent surface (withoutintermediate generation of spheroid or embryoid bodies). See, forexample, international patent application publication No. WO2017/072763, incorporated by reference herein in its entirety.

Thus, according to an aspect of the present disclosure, at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% of the cells which are subjected to directeddifferentiation on the adherent surface are undifferentiated ESCs andexpress markers of pluripotency. For example, at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the cells are Oct4⁺TRA− I-60⁺. The non-differentiatedESCs may express other markers of pluripotency, such as NANOG, Rex-1,alkaline phosphatase, Sox2, TDGF-beta, SSEA-3, SSEA-4, SSEA-5, OCT4,TRA-1-60 and/or TRA-1-81.

In one exemplary differentiation protocol, the non-differentiatedembryonic stem cells are differentiated towards the RPE cell lineage onan adherent surface using a first differentiating agent and then furtherdifferentiated towards RPE cells using a member of the transforminggrowth factor-β(TGFβ) superfamily, (e.g. TGF I, TGF2, and TGF 3subtypes, as well as homologous ligands including activin (e.g., activinA, activin B, and activin AB), nodal, anti-mullerian hormone (AMH), somebone morphogenetic proteins (BMP), e.g. BMP2, BMP3, BMP4, BMP5, BMP6,and BMP7, and growth and differentiation factors (GDF)). According to aspecific embodiment, the member of the transforming growthfactor-β(TGFβ) superfamily is activin A—e.g. between 20-200 ng/ml, e.g.100-180 ng/ml.

According to some embodiments, the first differentiating agent isnicotinamide (NA) used at concentrations of between about 1-100 mM, 5-50mM, 5-20 mM, and e.g. 10 mM. According to other embodiments, the firstdifferentiating agent is 3-aminobenzmine.

NA, also known as “niacinamide”, is the amide derivative form of VitaminB3 (niacin) which is thought to preserve and improve beta cell function.NA has the chemical formula C6H6N20. NA is essential for growth and theconversion of foods to energy, and it has been used in arthritistreatment and diabetes treatment and prevention.

According to some embodiments, the nicotinamide is a nicotinamidederivative or a nicotinamide mimic. The term “derivative of nicotinamide(NA)” as used herein denotes a compound which is a chemically modifiedderivative of the natural NA. In one embodiment, the chemicalmodification may be a substitution of the pyridine ring of the basic NAstructure (via the carbon or nitrogen member of the ring), via thenitrogen or the oxygen atoms of the amide moiety. When substituted, oneor more hydrogen atoms may be replaced by a substituent and/or asubstituent may be attached to a N atom to form a tetravalent positivelycharged nitrogen. Thus, the nicotinamide of the present inventionincludes a substituted or non-substituted nicotinamide. In anotherembodiment, the chemical modification may be a deletion or replacementof a single group, e.g. to form a thiobenzamide analog of NA, all ofwhich being as appreciated by those versed in organic chemistry. Thederivative in the context of the invention also includes the nucleosidederivative of NA (e.g. nicotinamide adenine). A variety of derivativesof NA are described, some also in connection with an inhibitory activityof the PDE4 enzyme (WO 03/068233; WO 02/060875; GB2327675A), or asVEGF-receptor tyrosine kinase inhibitors (WOO I/55114). For example, theprocess of preparing 4-aryl-nicotinamide derivatives (WO 05/014549).Other exemplary nicotinamide derivatives are disclosed in WOO I/55114and EP2128244.

Nicotinamide mimics include modified forms of nicotinamide, and chemicalanalogs of nicotinamide which recapitulate the effects of nicotinamidein the differentiation and maturation of RPE cells from pluripotentcells. Exemplary nicotinamide mimics include benzoic acid,3-aminobenzoic acid, and 6-aminonicotinamide. Another class of compoundsthat may act as nicotinamide mimics are inhibitors of poly(ADP-ribose)polymerase (PARP). Exemplary PARP inhibitors include 3-aminobenzamide,Iniparib (BSI 201), Olaparib (AZD-2281), Rucaparib (AG014699,PF-01367338), Veliparib (ABT-888), CEP 9722, MK 4827, and BMN-673.

Additional contemplated differentiation agents include for examplenoggin, antagonists of Wnt (Dkk1 or IWR1e), nodal antagonists (Lefty-A),retinoic acid, taurine, GSK3b inhibitor (CHIR99021) and notch inhibitor(DAPT).

According to certain embodiments, the differentiation is effected asfollows: (a) culture of ESCs in a medium comprising a firstdifferentiating agent (e.g. nicotinamide); and (b) culture of cellsobtained from step a) in a medium comprising a member of the TGFβsuperfamily (e.g. activin A) and the first differentiating agent (e.g.nicotinamide). Step (a) may be effected in the absence of the member ofthe TGFβ superfamily (e.g. activin A).

In some embodiments, the medium in step (a) is completely devoid of amember of the TGFβ superfamily. In other embodiments, the level of TGFβsuperfamily member in the medium is less than 20 ng/ml, 10 ng/ml, 1ng/ml or even less than 0.1 ng/ml.

The above described protocol may be continued by culturing the cellsobtained in step (b) in a medium comprising the first differentiatingagent (e.g. nicotinamide), but devoid of a member of the TGFβsuperfamily (e.g. activin A). This step is referred to herein as step(b*).

The above described protocol is now described in further detail, withadditional embodiments.

Step (a): The differentiation process is started once sufficientquantities of ESCs are obtained. They are typically removed from thecell culture (e.g. by using collagenase A, dispase, TrypLE select, EDTA)and plated onto a non-adherent substrate (e.g. cell culture plate suchas Hydrocell or an agarose-coated culture dish, or petri bacteriologicaldishes) in the presence of nicotinamide (and the absence of activin A).Exemplary concentrations of nicotinamide are between 0.01-100 mM,0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, and 10 mM. Once the cells areplated onto the non-adherent substrate (e.g. cell culture plate), thecell culture may be referred to as a cell suspension, preferablyfree-floating clusters in a suspension culture, i.e. aggregates of cellsderived from human embryonic stem cells (hESCs). The cell clusters donot adhere to any substrate (e.g., culture plate, carrier). Sources offree floating stem cells were previously described in WO 06/070370,which is herein incorporated by reference in its entirety. This stagemay be effected for a minimum of 1 day, more preferably two days, threedays, 1 week or even 14 days. Preferably, the cells are not cultured formore than 3 weeks in suspension together with the nicotinamide e.g.between 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10mM (and in the absence of activin A). In one embodiment, the cells arecultured for 6-8 days in suspension together with the nicotinamide e.g.,between 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10mM (and in the absence of activin A).

According to some embodiments, when the cells are cultured on thenon-adherent substrate e.g., cell culture plates, the atmospheric oxygenconditions are 20%. However, manipulation of the atmospheric oxygenconditions is also contemplated such that the atmospheric oxygen percentis less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% or even less than about5% (e.g., between 1%-20%, 1%-10% or 0-5%). According to otherembodiments, the cells are cultured on the non-adherent substrateinitially under normal atmospheric oxygen conditions and then lowered toless than normal atmospheric oxygen conditions. In some embodiments, thecells are cultured under lower oxygen levels during earlydifferentiation and then under higher oxygen levels during latedifferentiation.

Examples of non-adherent cell culture plates include those manufacturedby Nunc (e.g., Hydrocell Cat No. 174912), etc.

The clusters can comprise at least 50-500,000, 50-100,000, 50-50,000,50-10,000, 50-5000, 50-1000 cells. According to one embodiment, thecells in the clusters are not organized into layers and form irregularshapes. In one embodiment, the clusters are devoid of pluripotentembryonic stem cells. In another embodiment, the clusters comprise smallamounts of pluripotent embryonic stem cells (e.g. no more than 5%, or nomore than 3% (e.g. 0.01-2.7%) cells that co-express OCT4 and TRA-1-60 atthe protein level). Typically, the clusters comprise cells that havebeen partially differentiated under the influence of nicotinamide. Suchcells primarily express neural and retinal precursor markers such asPAX6, Rax, Six3 and/or CHX10.

The clusters may be dissociated using enzymatic or non-enzymatic methods(e.g., mechanical) known in the art. According to some embodiments, thecells are dissociated such that they are no longer in clusters—e.g.aggregates or clumps of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells,2-5000 cells, 2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells.According to a particular embodiment, the cells are in a single cellsuspension.

The cells (e.g., dissociated cells) can then be plated on an adherentsubstrate and cultured in the presence of nicotinamide e.g. between0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM (andthe absence of activin A). This stage may be effected for a minimum of 1day, more preferably two days, three days, 1 week or even 14 days.Preferably, the cells are not cultured for more than 3 weeks in thepresence of nicotinamide (and in the absence of activin). In anexemplary embodiment, this stage is effected for 6-7 days.

According to other embodiments, when the cells are cultured on theadherent substrate e.g., laminin, the atmospheric oxygen conditions are20%. They may be manipulated such that the percentage is less than about20%, 15%, 10%, more preferably less than about 9%, less than about 8%,less than about 7%, less than about 6% and more preferably about 5%(e.g., between 1%-20%, 1%-10% or 0-5%).

According to some embodiments, the cells are cultured on the adherentsubstrate initially under normal atmospheric oxygen conditions andsubsequently the oxygen is lowered to less than normal atmosphericoxygen conditions. According to other embodiments, the cells arecultured on the adherent substrate initially under lower than normalatmospheric oxygen conditions and subsequently the oxygen is raised tonormal atmospheric oxygen conditions.

Examples of adherent substrates or a mixture of substances could includebut are not limited to fibronectin, laminin, polyD-lysine, collagen andgelatin.

Step (b): Following the first stage of directed differentiation, (stepa; i.e. culture in the presence of nicotinamide (e.g., between 0.01-100mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM), thepartially-differentiated cells are then subjected to a further stage ofdifferentiation on an adherent substrate—culturing in the presence ofactivin A (e.g., 0.01-1000 ng/ml, 0.1-200 ng/ml, 1-200 ng/ml—for example140 ng/ml, 150 ng/ml, 160 ng/ml or 180 ng/ml). Thus, activin A may beadded at a final molarity of 0.1 pM-10 nM, 10 pM-10 nM, 0.1 nM-10 nM, 1nM-10 nM, for example 5.4 nM.

Nicotinamide may be added at this stage too (e.g., between 0.01-100 mM,0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM). This stage may beeffected for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks,one week to eight weeks, one week to four weeks, for example for atleast one day, at least two days, at least three days, at least 5 days,at least one week, at least 9 days, at least 10 days, at least twoweeks, at least three weeks, at least four weeks, at least five weeks,at least six weeks, at least seven weeks, at least eight weeks, at leastnine weeks, at least ten weeks.

According to some embodiments, this stage is effected for about eightdays to about two weeks. This stage of differentiation may be effectedat low or normal atmospheric oxygen conditions, as detailed hereinabove.

Step (b*): Following the second stage of directed differentiation (i.e.,culture in the presence of nicotinamide and activin A on an adherentsubstrate; step (b), the further differentiated cells are optionallysubjected to a subsequent stage of differentiation on the adherentsubstrate—culturing in the presence of nicotinamide (e.g., between0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM), inthe absence of activin A. This stage may be effected for at least oneday, 2, days, 5 days, at least one week, at least two weeks, at leastthree weeks or even four weeks. This stage of differentiation may alsobe carried out at low or normal atmospheric oxygen conditions, asdetailed herein above.

The basic medium in which the ESCs are differentiated is any known cellculture medium known in the art for supporting cells growth in vitro,typically, a medium comprising a defined base solution, which includessalts, sugars, amino acids and any other nutrients required for themaintenance of the cells in the culture in a viable state. According toa specific embodiment, the basic medium is not a conditioned medium.Non-limiting examples of commercially available basic media that may beutilized in accordance with the invention comprise NUTRISTEM® (withoutbFGF and TGF for ESC differentiation, with bFGF and TGF for ESCexpansion), NEUROBASAL™, KO- DMEM, DMEM, DMEM/FI2, CELLGRO™ Stem CellGrowth Medium, or X-VIVO™. The basic medium may be supplemented with avariety of agents as known in the art dealing with cell cultures. Thefollowing is a non-limiting reference to various supplements that may beincluded in the culture to be used in accordance with the presentdisclosure: serum or with a serum replacement containing medium, suchas, without being limited thereto, knock out serum replacement (KOSR),NUTRIDOMA-CS, TCH™, N2, N2 derivative, or B27 or a combination; anextracellular matrix (ECM) component, such as, without being limitedthereto, fibronectin, laminin, collagen and gelatin. The ECM may then beused to carry the one or more members of the TGFβ superfamily of growthfactors; an antibacterial agent, such as, without being limited thereto,penicillin and streptomycin; and non-essential amino acids (NEAA),neurotrophins which are known to play a role in promoting the survivalof SCs in culture, such as, without being limited thereto, BDNF, NT3,NT4.

According to some embodiments, the medium used for differentiating theESCs is NUTRISTEM® medium (Biological Industries, 06-5102-01-IA).

According to some embodiments, differentiation and expansion of ESCs areperformed under xeno free conditions.

According to other embodiments, the proliferation/growth medium isdevoid of xeno contaminants i.e., free of animal derived components suchas serum, animal derived growth factors and albumin. Thus, accordingthese embodiments, the culturing is performed in the absence of xenocontaminants.

Other methods for culturing ESCs under xeno free conditions are providedin U.S. Patent Application No. 20130196369, the contents of which areincorporated herein by reference in its entirety.

The preparations comprising RPE cells may be prepared in accordance withGood Manufacturing Practices (GMP) (e.g., the preparations areGMP-compliant) and/or current Good Tissue Practices (GTP) (e.g., thepreparations may be GTP-compliant).

During differentiation steps, the embryonic stem cells may be monitoredfor their differentiation state. Cell differentiation can be determinedupon examination of cell or tissue-specific markers which are known tobe indicative of differentiation.

Tissue/cell specific markers can be detected using immunologicaltechniques well known in the art [Thomson J A et al., (1998). Science282: 1145-7]. Examples include, but are not limited to, flow cytometryfor membrane-bound or intracellular markers, immunohistochemistry forextracellular and intracellular markers and enzymatic immunoassay, forsecreted molecular markers.

Following the stages of differentiation described herein above, a mixedcell population can be obtained comprising both pigmented andnon-pigmented cells. According to this aspect, the cells of the mixedcell population are removed from the plate. In some embodiments, this iseffected enzymatically (e.g., using trypsin, (TrypLE Select); see forexample, international patent application publication No. WO2017/021973, incorporated by reference herein in its entirety).According to this aspect of the present invention, at least 10%, 20%,30%, at least 40%, at least 50%, at least 60%, at least 70% of the cellswhich are removed from the culture (and subsequently expanded) arenon-pigmented cells. In other embodiments, this is effectedmechanically—e.g., using a cell scraper. In yet other embodiments, thisis effected chemically (e.g., EDTA). Combinations of enzymatic andchemical treatment are also contemplated. For example, EDTA andenzymatic treatments can be used. Furthermore, at least 10%, 20% or even30% of the cells which are removed from the culture (and subsequentlyexpanded) are pigmented cells.

According to this aspect of the present disclosure, at least 50%, 60%,70%, 80%, 90%, 95%, 100% of all the cells in the culture are removed(and subsequently expanded).

Expansion of the mixed population of cells may be effected on an extracellular matrix, e.g., gelatin, collagen I, collagen IV, laminin (e.g.,laminin 521), fibronectin and poly-D-lysine. For expansion, the cellsmay be cultured in serum-free KOM, serum comprising medium (e.g., DMEMwith 20% human serum) or NUTRISTEM® medium (06-5102-01-IA, BiologicalIndustries). Under these culture conditions, after passaging undersuitable conditions, the ratio of pigmented cells to non-pigmented cellsincreases such that a population of purified RPE cells is obtained. Suchcells show the characteristic polygonal shape morphology andpigmentation of RPE cells.

In one embodiment, the expanding is effected in the presence ofnicotinamide (e.g., between 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM,5-20 mM, e.g., 10 mM), and in the absence of activin A.

The mixed population of cells may be expanded in suspension (with orwithout a micro-carrier) or in a monolayer. The expansion of the mixedpopulation of cells in monolayer cultures or in suspension culture maybe modified to large scale expansion in bioreactors or multi/hyperstacks by methods well known to those versed in the art.

According to some embodiments, the expansion phase is effected for atleast one 20 weeks, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8weeks, at least 9 weeks or even 10 weeks. Preferably, the expansionphase is effected for 1 week-10 weeks, more preferably 2 weeks-10 weeks,more preferably, 3 weeks-10 weeks, more preferably 4 weeks-10 weeks, or4 weeks-8 weeks.

According to still other embodiments, the mixed population of cells arepassaged at least 1 time during the expansion phase, at least twiceduring the expansion phase, at least three times during the expansionphase, at least four times during the expansion phase, at least fivetimes during the expansion phase, or at least six times during theexpansion phase.

The present inventors have shown that when cells are collectedenzymatically, it is possible to continue the expansion for more than 8passages, more than 9 passages and even more than 10 passages (e.g.,11-15 passages). The number of total cell doublings can be increased togreater than 30, e.g., 31, 32, 33, 34 or more. (See international patentapplication publication number WO 2017/021973, incorporated herein byreference in its entirety).

The population of RPE cells generated according to the methods describedherein may be characterized according to a number of differentparameters. Thus, for example, the RPE cells obtained may be polygonalin shape and pigmented.

It will be appreciated that the cell populations disclosed herein aregenerally devoid of undifferentiated human embryonic stem cells.According to some embodiments, less than 1:250,000 cells areOct4+TRA-1-60+ cells, as measured for example by FACS. The cells mayalso have down regulated (by more than 5,000 fold) expression of GDF3 orTDGF as measured by PCR. The RPE cells of this aspect, do not expressembryonic stem cell markers. Said one or more embryonic stem cellmarkers may comprise OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2,TDGF-beta, SSEA-3, SSEA-4, TRA-1-60, and/or TRA-1-81.

The therapeutic RPE cell preparations may be substantially purified,with respect to non-RPE cells, comprising at least about 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells. Thetherapeutic RPE cell preparation may be essentially free of non-RPEcells or consist of RPE cells. For example, the substantially purifiedpreparation of RPE cells may comprise less than about 25%, 20%, 15%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% non-RPE cell type. Forexample, the RPE cell preparation may comprise less than about 25%, 20%,15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%,0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%,0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%,0.0004%, 0.0003%, 0.0002%, or 0.0001% non-RPE cells.

The RPE cell preparations may be substantially pure, both with respectto non-RPE cells and with respect to RPE cells of other levels ofmaturity. The preparations may be substantially purified, with respectto non-RPE cells, and enriched for mature RPE cells. For example, in RPEcell preparations enriched for mature RPE cells, at least about 30%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% of the RPE cells are matureRPE cells. The preparations may be substantially purified, with respectto non-RPE cells, and enriched for differentiated RPE cells rather thanmature RPE cells. For example, at least about 30%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the RPE cells may be differentiated RPE cellsrather than mature RPE cells.

The preparations described herein may be substantially free ofbacterial, viral, or fungal contamination or infection, including butnot limited to the presence of HIV I, HIV 2, HBV, HCV, HAV, CMV, HTLV 1,HTLV 2, parvovirus B19, Epstein-Barr virus, or herpesvirus 1 and 2,SV40, HHV5, 6, 7, 8, CMV, polyoma virus, HPV, Enterovirus. Thepreparations described herein may be substantially free of mycoplasmacontamination or infection.

Another way of characterizing the cell populations disclosed herein isby marker expression. Thus, for example, at least 50%, 60% 70%, 80%,85%, 90%, 95% or 100% of the cells may express Bestrophin 1, as measuredby immunostaining. According to one embodiment, between 80-100% of thecells express bestrophin 1.

According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%,97% or 100% of the cells express Microphthalmia-associated transcriptionfactor (MITF), as measured by immunostaining. For example, between80-100% of the cells express MITF.

According to other embodiments, at least 50%, 60% 70%, 80%, 85%, 87%,89%, 90%, 95%, 97% or 100% of the cells express bothMicrophthalmia-associated transcription factor (MITF) and bestrophin 1,as measured by immunostaining. For example, between 80-100% of the cellsco-express MITF and bestrophin 1.

According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%,97% or 100% of the cells express both Microphthalmia-associatedtranscription factor (MITF) and ZO-1, as measured by immunostaining. Forexample, between 80-100% of the cells co-express MITF and ZO-1.

According to other embodiments, at least 50%, 60% 70%, 80%, 85%, 87%,89%, 90%, 95%, 97% or 100% of the cells express both ZO-1 and bestrophin1, as measured by immunostaining. For example, between 80-100% of thecells co-express ZO-1 and bestrophin 1.

According to another embodiment, at least 50%, 60% 70% 80%, 85%, 87%,89%, 90%, 95%, 97% or 100% of the cells express paired box gene 6(PAX-6) as measured by immunostaining or FACS.

According to another embodiment, at least 50%, 60% 70%, 80%, 85%, 87%,89%, 90%, 95%, 97% or 100% of the cells express cellular retinaldehydebinding protein (CRALBP), as measured by immunostaining. For example,between 85-100% of the cells express CRALBP.

According to another embodiment, at least 50%, 60% 70%, 80%, 85%, 87%,89%, 90%, 95%, 97% or 100% of the cells express cellular MelanocytesLineage-Specific Antigen GP100 (PMEL17), as measured by immunostaining.For example, between 85-100% of the cells express PMEL17.

The RPE cells typically co-express markers indicative of terminaldifferentiation, e.g. bestrophin 1, CRALBP and/or RPE65. In addition,the RPE cells described herein may express markers for RPE primarycilia, such as ARL13B and GT335.

Following the expansion phase, cell populations comprising RPE cells areobtained whereby at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% thereof are CRALBP+ PMEL17-+.

In certain embodiments, RPE cell compositions may be produced accordingto the following methods: (1) culturing hESCs on hUCFs in center well(CW) plates for 2 weeks in NUT+ with human serum albumin (HSA), (2)mechanical passaging to expand the hESCs on hUCFs in CW plates forbetween four to five weeks (or until desired amount of cells) in NUT+with HSA, (3) continue to expand hESC colonies (using for example,collagenase) on hUCFs in 6 cm plates for an additional week in NUT+ withHSA, (4) prepare spheroid bodies (SB) by transferring colonies fromabout five 6 cm plates into 1 HydroCell for about one week in NUT− withnicotinamide (NIC), (5) flattening of SBs on Lam511 may be carried outby transferring the SBs to 2-3 wells of a 6-well plate for about oneweek in NUT− with NIC, (6) culture adherent cells on Lam511 in NUT− withNIC and Activin for about one to two weeks and replace media with NUT−with NIC and culture for between one and three weeks, (7) enrich forpigmented cells using enzymes, such as TrypLE Select for example, (8)expand RPE cells on gelatin in flasks for between about two to nineweeks (replacing media) in 20% human serum and NUT−, and (9) harvest RPEcells.

Harvesting of the expanded population of RPE cells may be carried outusing methods known in the art (e.g. using an enzyme such as trypsin, orchemically using EDTA, etc). In some embodiments, the RPE cells may bewashed using an appropriate solution, such as PBS or BSS plus. In someembodiments, an enzyme neutralizing solution may be used subsequent toharvesting or enriching for RPE cells. The neutralizing solution maycomprise for example, medium with or without human serum or human serumalbumin. In some embodiments, prolonged incubation in enzymeneutralizing solution with or without HS or HAS has no effect on cellviability or cell recovery.

In other embodiments, the RPE cells may be filtered prior to formulationof the RPE cells for cryopreservation and administration to a subjectdirectly after thawing. In some embodiments, the percent viability ofpost-filtered cells is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the percentviability of post-filtered cells stored in a neutralization solution forbetween about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Following harvesting, the expanded population of RPE cells can beformulated at a specific therapeutic dose (e.g., number of cells) andcryopreserved for shipping to the clinic. The ready to administer (RTA)RPE cell therapy composition can then be administered directly afterthawing without further processing. Examples of media suitable forcryopreservation include but are not limited to 90% Human Serum/10%DMSO, Media 3 10% (CS10), Media 2 5% (CS5) and Media 1 2% (CS2), StemCell Banker, PRIME XV® FREEZIS, HYPOTHERMASOL®, CSB, Trehalose, etc.

In some embodiments, the percent viability of post-filtered cells storedin a cryopreservation medium for between about 0 to about 8 hours is atleast about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%. In other embodiments, the percent recovery ofpost-filtered cells stored in a cryopreservation medium for betweenabout 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In further embodiments, the percent viability of post-filtered cellsstored in a neutralization medium for between about 0 to about 8 hoursfollowed by storage in cryopreservation medium for between about 0 toabout 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the percentrecovery of post-filtered cells stored in a neutralization medium forbetween about 0 to about 8 hours followed by storage in cryopreservationmedium for between about 0 to about 8 hours is at least about 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In yet other embodiments, the percent viability of post-filtered cellsstored in a neutralization medium for between about 0 to about 8 hoursfollowed by storage in cryopreservation medium for between about 0 toabout 8 hours, post-thawing of the cryopreserved composition, is atleast about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%. In still other embodiments, the percent recovery ofpost-filtered cells stored in a neutralization medium for between about0 to about 8 hours followed by storage in cryopreservation medium forbetween about 0 to about 8 hours, post-thawing of the cryopreservedcomposition, is at least about, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, post-filtered RPE cells stored in a neutralizationmedium for between about 0 to about 8 hours followed by storage incryopreservation medium for between about 0 to about 8 hours,post-thawing of the cryopreserved composition are capable of secretingPEDF at between about 1,500 ng/ml/day to about 4,500 ng/ml/day, about2,000 ng/ml/day to about 3,000 ng/ml/day. In other embodiments,post-filtered RPE cells stored in a neutralization medium for betweenabout 0 to about 8 hours followed by storage in cryopreservation mediumfor between about 0 to about 8 hours, post-thawing of the cryopreservedcomposition are capable of being expanded to at least between about1.2×10⁶ and 5×10⁶, or about 2.5× ×10⁶ to about 4×10⁶ cells in 14 days.

In some embodiments, the percent viability of post-filtered RPE cellsstored in a neutralization medium for between about 0 to about 8 hoursat room temperature is at least about, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments,the percent viability of post-filtered RPE cells stored in acryopreservation medium for between about 0 to about 8 hours at roomtemperature is at least about, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%. In further embodiments, thepercent viability of post-filtered cells stored in a neutralizationsolution at room temperature for between about 0 to about 8 hoursfollowed by storage in cryopreservation medium for between about 0 toabout 8 hours at room temperature is at least about 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In still furtherembodiments, the percent recovery of post-filtered cells stored in aneutralization solution at room temperature for between about 0 to about8 hours followed by storage in cryopreservation medium for between about0 to about 8 hours at room temperature is at least about 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%,115%, 120%, 125%, 130%, 140%, 150%.

RPE cells formulated in cryopreservation media appropriate for post thawready to administer (RTA) applications may comprise RPE cells suspendedin adenosine, dextran-40, lactobionic acid, HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione,potassium chloride, potassium bicarbonate, potassium phosphate,dextrose, sucrose, mannitol, calcium chloride, magnesium chloride,potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), andwater. An example of this cryopreservation media is availablecommercially under the tradename, CRYOSTOR® and is manufactured byBioLife Solutions, Inc.

DMSO can be used as a cryoprotective agent to prevent the formation ofice crystals, which can kill cells during the cryopreservation process.In some embodiments, the cryopreservable RPE cell therapy compositioncomprises between about 0.1% and about 2% DMSO (v/v). In someembodiments, the RTA RPE cell therapy composition comprises betweenabout 1% and about 20% DMSO. In some embodiments, the RTA RPE celltherapy composition comprises about 2% DMSO. In some embodiments, theRTA RPE cell therapy composition comprises about 5% DMSO.

In some embodiments, RPE cell therapies formulated in cryopreservationmedia appropriate for post thaw ready to administer (RTA) applicationsmay comprise RPE cells suspended in cryopreservation media that does notcontain DMSO. For example, RTA RPE therapeutic cell compositions maycomprise RPE cells suspended in Trolox, Na+, K+, Ca2+, Mg2+, c1−,H2P04−, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40,adenosine, glutathione without DMSO (dimethyl sulfoxide, (CH₃)₂SO) orany other dipolar aprotic solvents. An example of this cryopreservationmedia is available commercially under the tradename, HYPOTHERMOSOL® orHYPOTHERMOSOL®-FRS and is also manufactured by BioLife Solutions, Inc.In other embodiments, RPE cell compositions formulated incryopreservation media appropriate for post thaw ready to administerapplications may comprise RPE cells suspended in Trehalose.

The RTA RPE cell therapy composition may optionally comprise additionalfactors that support RPE engraftment, integration, survival, potency,etc. In some embodiments, the RTA RPE cell therapy composition comprisesactivators of function of the RPE cell preparations described herein. Insome embodiments, the RTA RPE cell therapy composition comprisesnicotinamide. In some embodiments, the RTA RPE cell therapy compositioncomprises nicotinamide at a concentration of between about 0.01-100 mM,0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM. In otherembodiments, the RTA RPE cell therapy composition comprises retinoicacid. In some embodiments, the RTA RPE cell therapy compositioncomprises retinoic acid at a concentration of between about 0.01-100 mM,0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM.

In some embodiments, the RTA RPE cell therapy composition may beformulated to include activators of various integrins that have beenshown to increase the adherence of the RPE cell preparations, such asthose described herein, to the Brunch's membrane. For example, in someembodiments, the RTA RPE cell therapy composition comprisesextracellular manganese (Mn2+) at a concentration of between about 5 μMand 1,000 μM. In other embodiments, the RTA RPE cell therapy compositioncomprises the conformation-specific monoclonal antibody, TS2/16.

In other embodiments, the RTA RPE cell therapy composition may also beformulated to include activators of RPE cell immune regulatory activity.

In some embodiments, the RTA RPE cell therapy composition may include aROCK inhibitor.

In some embodiments, the RTA RPE cell therapy composition may beformulated in a medium comprising components that decrease the molecularcell stress during freezing and thawing processes by scavenging of freeradicals, pH buffering, oncotic/osmotic support and maintenance of theionic concentration balance.

In some embodiments, RPE cell therapies formulated in cryopreservationmedia appropriate for post thaw ready to administer applications maycomprise one or more immunosuppressive compounds. In certainembodiments, RPE cell therapies formulated in cryopreservation mediaappropriate for post thaw ready to administer applications may compriseone or more immunosuppressive compounds that are formulated for slowrelease of the one or more immunosuppressive compounds.Immunosuppressive compounds for use with the formulations describedherein may belong to the following classes of immunosuppressive drugs:Glucocorticoids, Cytostatics (e.g. alkylating agent or antimetabolite),antibodies (polyclonal or monoclonal), drugs acting on immunophilins(e.g., cyclosporin, Tacrolimus or Sirolimus). Additional drugs includeinterferons, opioids, TNF binding proteins, mycophenolate and smallbiological agents. Examples of immunosuppressive drugs include:mesenchymal stem cells, anti-lymphocyte globulin (ALG) polyclonalantibody, anti-thymocyte globulin (ATG) polyclonal antibody,azathioprine, BAS 1 LI X IMAB® (anti-I L-2Ra receptor antibody),cyclosporin (cyclosporin A), DACLIZUMAB® (anti-I L-2Ra receptorantibody), everolimus, mycophenolic acid, RITUX IMAB® (anti-CD20antibody), sirolimus, tacrolimus, Tacrolimus and or Mycophenolatemofetil.

The number of viable cells that may be administered to the subject aretypically between at least about 50,000 and about 5×10⁶ per dose. Insome embodiments, the RTA RPE cell therapy composition comprises atleast 100,000 viable cells. In some embodiments, the RTA RPE celltherapy composition comprises at least 150,000 viable cells. In someembodiments, the RTA RPE cell therapy composition comprises at least200,000 viable cells. In some embodiments, the RTA RPE cell therapycomposition comprises at least 250,000 viable cells. In someembodiments, the RTA RPE cell therapy composition comprises at least300,000 viable cells. In some embodiments, the RTA RPE cell therapycomposition comprises at least 350,000 viable cells. In someembodiments, the RTA RPE cell therapy composition comprises at least400,000 viable cells. In some embodiments, the RTA RPE cell therapycomposition comprises at least 450,000 viable cells. In someembodiments, the RTA RPE cell therapy composition comprises at least500,000 viable cells. In some embodiments, the RTA RPE cell therapycomposition comprises at least 600,000, at least 700,000, at least800,000, at least 900,000, at least 1,000,000, at least, 2,000,000, atleast 3,000,000, at least, 4,000,000, at least 5,000,000 at least6,000,000, at least 7,000,000, at least 8,000,000, at least 9,000,000,at least 10,000,000, at least 11,000,000, or at least 12,000,000 viablecells.

In some embodiments, the volume of the RTA RPE formulation administeredto the subject is between about 50 μl to about 100 μl, about 25 μl toabout 100 μl, about 100 μl to about 150 ul, or about 10 μl to about 200μl. In certain embodiments, two doses of between 10 μl and 200 μl of theRTA RPE formulation can be administered. In certain embodiments, thevolume of RTA RPE formulation is administered to the subretinal space ofa subject's eye. In certain embodiments, the subretinal delivery methodcan be transvitreal or suprachoroidal. In some embodiments, the volumeof RTA RPE formulation can be injected into the subject's eye.

In certain embodiments, the RTA RPE therapeutic cell compositions may beformulated at a cell concentration of between about 100,000 cells/ml toabout 1,000,000 cells/ml. In certain embodiments, the RTA RPE celltherapy may be formulated at a cell concentration of about 1,000,000cells/ml, about 2,000,000 cells/ml, about 3,000,000 cells/ml, about4,000,000 cells/ml, about 5,000,000 cells/ml, 6,000,000 cells/ml,7,000,000 cells/ml, 8,000,000 cells/ml, about 9,000,000 cells/ml, about10,000,000 cells/ml, about 11,000,000 cells/ml, about 12,000,000cells/ml, 13,000,000 cells/ml, 14,000,000 cells/ml, 15,000,000 cells/ml,16,000,000 cells/ml, about 17,000,000 cells/ml, about 18,000,000cells/ml, about 19,000,000 cells/ml, or about 20,000,000 cells/ml.

In some embodiments, the RTA RPE cell therapy composition may becryopreserved and stored at a temperature of between about −4° C. toabout −200° C. In some embodiments, the RTA RPE cell therapy compositionmay be cryopreserved and stored at a temperature of between about −20°C. to about −200° C. In some embodiments, the RTA RPE cell therapycomposition may be cryopreserved and stored at a temperature of betweenabout −70° C. to about −196° C. In some embodiments, the temperatureadequate for cryopreservation or a cryopreservation temperature,comprises a temperature of between about −4° C. to about −200° C., or atemperature of between about −20° C. to about −200° C., −70° C. to about−196° C. In some embodiments, the RTA RPE cell therapy composition maybe stored frozen for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days. In other embodiments, the RPE cells may be stored frozen forbetween about 1.5 to 48 months. In other embodiments, the RTA RPE celltherapy composition may be stored frozen for between about 1 to about 48months without a decrease in percent viability or cell recovery. In someembodiments, the RTA RPE cell therapy composition may be stored for atleast about 38 hours at 2-8° C., while maintaining stability.

In some embodiments, the RTA RPE cell therapy composition may be shippedfrozen over 8,000 miles without a decrease in percent viability, percentcell recovery, or potency.

It would be well appreciated by those versed in the art that thederivation of RPE cells is of great benefit. They may be used as an invitro model for the development of new drugs to promote their survival,regeneration and function. RPE cells may serve for high throughputscreening for compounds that have a toxic or regenerative effect on RPEcells. They may be used to uncover mechanisms, new genes, soluble ormembrane-bound factors that are important for the development,differentiation, maintenance, survival and function of photoreceptorcells.

The RPE described herein cells may also serve as an unlimited source ofRPE cells for transplantation, replenishment and support ofmalfunctioning or degenerated RPE cells in retinal degenerations andother degenerative disorders. Furthermore, genetically modified RPEcells may serve as a vector to carry and express genes in the eye andretina after transplantation.

Eye conditions for which the RPE cells may serve as therapeuticsinclude, but are not limited to retinal diseases or disorders generallyassociated with retinal dysfunction, retinal injury, and/or loss ofretinal pigment epithelium. A non-limiting list of conditions which maybe treated in accordance with the invention comprises retinitispigmentosa, lebers congenital amaurosis, hereditary or acquired maculardegeneration, age related macular degeneration (AMD), non-exudative(dry) AMD, Geographic Atrophy (GA), Best disease, retinal detachment,gyrate atrophy, choroideremia, pattern dystrophy as well as otherdystrophies of the RPE, Stargardt disease, RPE and retinal damage due todamage caused by any one of photic, laser, inflammatory, infectious,radiation, neo vascular or traumatic injury.

Exemplary degenerative disorders that may be treated using the cells ofthis aspect of the present invention include neurodegenerative disordersincluding but not limited to Parkinson's, ALS, Multiple Sclerosis,Huntingdon's disease, autoimmune encephalomyelitis, diabetic neuropathy,Alzheimer's and epilepsy.

Subjects which may be treated include primate (including humans),canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)),avian, and other subjects. Humans and non-human animals havingcommercial importance (e.g., livestock and domesticated animals) are ofparticular interest. Exemplary mammals which may be treated include,canines; felines; equines; bovines; ovines; rodentia, etc. and primates,particularly humans. Non-human animal models, particularly mammals, e.g.primate, murine, lagomorpha, etc. may be used for experimentalinvestigations.

The RPE cells generated as described herein may be transplanted tovarious target sites within a subject's eye or other locations (forexample in the brain). In accordance with one embodiment, thetransplantation of the RPE cells is to the subretinal space of the eye,which is the normal anatomical location of the RPE (between thephotoreceptor outer segments and the choroid). In addition, dependentupon migratory ability and/or positive paracrine effects of the cells,transplantation into additional ocular compartments can be consideredincluding but not limited to the vitreal space, inner or outer retina,the retinal periphery and within the choroids.

The transplantation may be performed by various techniques known in theart. Methods for performing RPE transplants are described in, forexample, U.S. Pat. Nos. 5,962,027, 6,045,791, and 5,941,250 and in EyeGraefes Arch Clin Exp Opthalmol March 1997; 235(3):149-58; BiochemBiophys Res Commun Feb. 24, 2000; 268(3): 842-6; Opthalmic Surg February1991; 22(2): 102-8. Methods for performing corneal transplants aredescribed in, for example, U.S. Pat. No. 5,755,785, and in Eye 1995; 9(Pt 6 Su): 6-12; Curr Opin Opthalmol August 1992; 3 (4): 473-81;Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; Ophthalmology April2000; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999;43(6): 502-8. If mainly paracrine effects are to be utilized, cells mayalso be delivered and maintained in the eye encapsulated within asemi-permeable container, which will also decrease exposure of the cellsto the host immune system (Neurotech USA CNTF delivery system; PNAS Mar.7, 2006 vol. 103(10) 3896-3901).

The step of administering may comprise intraocular administration of theRPE cells into an eye in need thereof. The intraocular administrationmay comprise injection of the RPE cells into the subretinal space.

In accordance with one embodiment, transplantation is performed via parsplana vitrectomy surgery followed by delivery of the cells through asmall retinal opening into the sub-retinal space or by direct injection.

In certain embodiments, administration may comprise a vitrectomyfollowed by delivery of the RTA therapeutic cell composition into thesubretinal space in the macular area via a cannula through a smallretinotomy. A total volume of 50-100 μL cell suspension, depending onthe cell dose can be implanted in areas at potential risk for GAexpansion.

In some embodiments, a single surgical procedure is performed in whichthe RTA therapeutic cell composition is delivered through a smallretinotomy, following vitrectomy, into a subretinal space created in themacular area, along the border between areas of GA, if present, and thebetter preserved extra-foveal retina and RPE layer. After the placementof a lid speculum, a standard 3-port vitrectomy can be performed. Thismay include the placement of a 23G or 25G infusion cannula and two 23Gor 25/23G ports (trocars). A core vitrectomy can then be performed with23G or 25G instruments, followed by detachment of the posterior vitreousface. The RTA therapeutic cell composition may be injected into thesubretinal space at a predetermined site within the posterior pole,preferably penetrating the retina in an area that is still relativelypreserved close to the border of GA, if present.

In some embodiments, the cell composition is administered by asuprachoroidal injection.

The RPE cells may be transplanted in various forms. For example, the RPEcells may be introduced into the target site in the form of single cellsuspension, with matrix or adhered onto a matrix or a membrane,extracellular matrix or substrate such as a biodegradable polymer or acombination. The RPE cells may also be printed onto a matrix orscaffold. The RPE cells may also be transplanted together(co-transplantation) with other retinal cells, such as withphotoreceptors. The effectiveness of treatment may be assessed bydifferent measures of visual and ocular function and structure,including, among others, best corrected visual acuity (BCVA), retinalsensitivity to light as measured by perimetry or microperimetry in thedark and light-adapted states, full-field, multi-focal, focal or patternelectroretinography 5 ERG), contrast sensitivity, reading speed, colorvision, clinical biomicroscopic examination, fundus photography, opticalcoherence tomography (OCT), fundus auto-fluorescence (FAF), infrared andmulticolor imaging, fluorescein or ICG angiography, adoptive optics andadditional means used to evaluate visual function and ocular structure.

The subject may be administered corticosteroids prior to or concurrentlywith the administration of the RPE cells, such as prednisolone ormethylprednisolone, Predforte. According to another embodiment, thesubject 1s not administered corticosteroids prior to or concurrentlywith the administration of the RPE cells, such as prednisolone ormethylprednisolone, Predforte.

Immunosuppressive drugs may be administered to the subject prior to,concurrently with and/or following treatment. The immunosuppressive drugmay belong to the following classes: Glucocorticoids, Cytostatics (e.g.alkylating agent or antimetabolite), antibodies (polyclonal ormonoclonal), drugs acting on immunophilins (e.g. cyclosporin, Tacrolimusor Sirolimus). Additional drugs include interferons, opioids, TNFbinding proteins, mycophenolate and small biological agents. Examples ofimmunosuppressive drugs include: mesenchymal stem cells, anti-lymphocyteglobulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG)polyclonal antibody, azathioprine, BAS 1LI X IMAB® (anti-I L-2Rareceptor antibody), cyclosporin (cyclosporin A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX IMAB®(anti-CD20 antibody), sirolimus, tacrolimus, Tacrolimus and orMycophenolate mofetil.

Alternatively, the RTA RPE cell therapy composition may be administeredwithout the use of immunosuppressive drugs.

Antibiotics may be administered to the subject prior to, concurrentlywith and/or following treatment. Examples of antibiotics include Oflox,Gentamicin, Chloramphenicol, Tobrex, Vigamox or any other topicalantibiotic preparation authorized for ocular use.

RTA RPE cell therapies formulated according to the present disclosure donot require the use of GMP facilities for preparation of the final doseformulation prior to injection into a subject's eye. The RTA RPE celltherapy formulations described herein may be cryopreserved in anon-toxic cryosolution that comprises the final dose formulation whichcan be shipped directly to the clinical site. When needed, theformulation can be thawed and administered into the subject's eyewithout having to perform any intermediate preparation steps.

RPE cells are involved in many processes critical for photoreceptorsurvival, including nutrient, water, and ion transport, lightabsorption, phagocytosis of shed photoreceptor outer segments (POS),re-isomerization of all-trans-retinal into 11-cis-retinal, which iscrucial for the visual cycle, immune regulation, secretion of essentialfactors, and formation of the blood-retinal barrier. As shown in FIG. 15, the RPE monolayer acts as a polarized metabolic gatekeeper between thePRs and the choroicapillaries (CC). The RPE has an apical to basolateralstructural and functional polarity. On the apical side, RPE cells formmultiple villi enabling direct contact with the POS and transportmolecules such as glucose and vitamin A from the choroicapillaries toPRs. On the basal side, RPE cells transport metabolites such as C02,lactate and water to the choroicapillaries and generate the underlyingbasal Bruch's membrane (BM) that separates the RPE from the choroidgenerating the blood-retinal barrier. On the lateral walls, adjoiningRPE cells form tight junctions. Barrier function can be used todetermine the potency of RPE cell cultures by measuring the tightjunctions formed between the cells. RPE tight junctions limitparacellular movement of ions and water across the RPE monolayer andmaintain the correct apico-basal distribution of RPE transporters. TheRPE cell compositions disclosed herein display barrier functiondetermined by the ability to generate Trans Epithelial ElectricalResistance (TEER) above 100Ω.

In addition, RPE cells secrete a variety of neurotrophic factors, suchas fibroblast growth factors (bFGF and aFGF), ciliary neurotrophicfactor (CNTF), pigment epithelium-derived factor (PEDF), brain-derivedneurotrophic factor (BDNF), vascular endothelial growth factor (VEGF)and others, that help to maintain the structural integrity ofchoriocapillaris endothelium and photoreceptors. RPE cells also secreteanti-inflammatory cytokines such as transforming growth factor (TGF)-β,important in establishing the immune privileged properties of the eye.The RPE cells used in the RTA therapeutic cell compositions describedherein are capable of secreting neurotrophic factors. The RPE cellcompositions disclosed herein also demonstrate polarized PEDF and VEGFsecretion which enhances RPE growth and blood vessel formation,respectively.

Different cell culture media can have an effect on the expansionefficiency of cells. However, the RPE cell compositions disclosed hereindemonstrate the ability to expand after being suspended in mediaformulations comprising DMSO.

The RPE cell compositions disclosed herein also display a percentage ofviable post thawed cells that allows the formulations to be used as aready to inject cell therapy, without the need to remove dead cells. Thepercent yield of the RPE cell compositions disclosed herein, as measuredby cells per milliliter, is characteristic of formulations that areoptimized to meet large scale clinical use requirements.

Example 1

Used herein are cell suspensions of RPE cells, derived from humanembryonic stem cells (hESCs) through a process of directeddifferentiation under xeno-free, GMP manufacturing conditions. Thesecells were expanded on irradiated human umbilical cord fibroblastfeeders (hUCFs). The expanded hESCs were then differentiated intoretinal pigment epithelium (RPE) cells using Nicotinamide and Activin A.The RPE cells were then expanded and cryopreserved in cryopreservationmedium.

Xeno-free GMP grade HAD-C I02 hESCs were expanded as colonies onirradiated xeno-free GMP-grade CRD008 hUCFs that were seeded onrecombinant human vitronectin (rhVTN) or on recombinant human Gelatin(rhGelatin). hESC expansion was carried out in the presence ofNUTRISTEM® medium that contains human serum albumin in addition to thegrowth factors basic FGF and TGF beta (Biological Industries). ExpandedhESCs were then transferred to a suspension culture to initiatedifferentiation in a directed manner under normal (atmospheric) 02conditions.

Spheroid bodies (SBs) were formed and then plated as an adherent cellculture under continued directed differentiation conditions towards aneural fate and subsequently towards an RPE cell fate.

At the end of the differentiation phase, cells were harvested using thefollowing two techniques and expanded 1) Non-pigmented areas weremanually excised and removed and the remaining pigmented cell areas wereenzymatically collected, and 2) Cells (pigmented and non-pigmented) werecollected enzymatically. Cells were then seeded and expanded for 3passages on top of rhGelatin covered cell culture plates according tomanufacturing instructions in the presence and absence of nicotinamideor on top of Laminin 521, Fibronectin, Collagen I or Collagen IV. Cellswere harvested and cryopreserved at passage 2 (P2) in cryo-mediumcomprised of 90% human serum and 10% DMSO, and in serum free xeno-freeGMP grade cryomedia (Media 2 (CS5) and Media 1 (CS2), BioLifeSolutions).

Example 2

Post thaw vitality and viability were assessed for therapeutic RPE cellscryopreserved in cryopreservation media with 5% dimethyl sulfoxide(DMSO) (Media 2, CS5) at cell densities of 1.5×10⁶ and 5×10⁶. Resultswere compared to the results of cells that were cryopreserved in 90%human serum (HS) with 10% DMSO using a controlled freezing machine(e.g., an isopropanol containing slow cooling apparatus). After thawingof 3 vials of each composition frozen in each cryopreservation media,viability was tested using a cell counter. Cells of each vial were thenseeded in a 12-well plate, at a density of 0.5×10⁶ viable cells/well ina final volume of 2 mL DMEM containing 20% human serum per well, for 24hours at 37° C. and 5% CO₂. At the end of the incubation period,cultures were washed with PBS. Following TrypLE Select treatment, cellswere enumerated using a cell counter. Percent vitality was thencalculated by dividing the average number of viable adhered cells withthe total number of seeded cells per well and multiplying the result by100.

As shown in FIG. 1 , the results demonstrate that RPE cells that werecryopreserved in the media used herein had similar post thaw viabilityand better post thaw vitality (better % cell adherence 24 hours postthawing) when compared to the cryopreservation medium comprised of 90%human serum and 10% DMSO (HS/DMSO).

Example 3

Therapeutic RPE cell compositions were formulated using xeno-freeGMP-grade reagents, xeno-free GMP-grade cells (HAD-C I02-hESCs grown onirradiated CRD008), as described in Example 1.

Assessment of CRALBP⁺PMEL17⁺ cells for measurement of RPE purity wasperformed at the end of the differentiation phase. As shown in Table 1αand Table 1 b, purity of RPE cells was at least 98.76% or greater forall RTA RPE cell therapy compositions formulated with CS2 (Media 1) orCS5 (Media 2).

Tight junctions generated between RPE cells enable the generation of theblood-retinal barrier and a polarized PEDF and VEGF secretion. PEDF issecreted to the apical side where it acts as an anti-angiogenic andneurotropic growth factor. VEGF is mainly secreted to the basal side,where it acts as a proangiogenic growth factor on the choroidalendothelium. RPE polarization (barrier function and polarized PEDF andVEGF secretion) was measured in a transwell system in cells at the endof the production process. As shown in Table 1a and 1 b, barrierfunction/trans-epithelial electrical resistance (TEER) was demonstratedas well as polarized secretion of PEDF and VEGF.

Control samples (Ctrl) were cryopreserved in 10% DMSO and 90% humanserum.

TABLE 1a Characterization of Therapeutic RPE Cells Cryopreserved inMedia 1 (CS2) and Media 2 (CS5) for Production Runs (PR) 1 and 2Therapeutic RPE PR 1 Therapeutic RPE PR 2 Mean ± SD (n) Mean ± SD (n)Test (Method) Ctrl CS2 CS5 Ctrl CS2 CS5 % Viability 85 ± 3 87 ± 1 89 ± 384 84 92 (Cell Counter) (n = 3) (n = 3) (n = 3) (n = 2) (n = 2) (n = 2)Purity (FACS): 99.78 99.54 99.57 98.74 99.50 99.87 % CRALBP⁺ PMEL17⁺Cells Potency: Transepithelial 274 175 225 663 739 753 ElectricalResistance (TEER) Polarized 3.6 3.2 4.1 4.0 6.6 6.1 PEDF Secretion(Apical to Basal Ratio) Polarized 1.4 1.5 1.7 3.7 2.3 2.2 VEGF Secretion(Basal to Apical Ratio)

TABLE 1b Characterization of Therapeutic RPE Cells Cryopreserved inMedia 1 (CS2) and Media 2 (CS5) for Experiments (PR) 3 and 4 TherapeuticRPE® PR 3 Therapeutic RPE® PR 4 Mean ± SD (n) Mean ± SD (n) Test(Method) Ctrl CS2 CS5 Ctrl CS2 CS5 % Viability 87 ± 5 89 ± 5 90 ± 4 8484 91 (Cell Counter) (n = 4) (n = 4) (n = 4) ( n= 2) (n = 2) (n = 2)Purity (FACS): 98.51 99.21 98.76 99.27 99.28 98.97 % CRALBP⁺ PMEL17⁺Cells Potency: Transepithelial NA 233 385 881 846 701 ElectricalResistance (TEER) Polarized NA 5.6 11.5 8.3 7.4 6.1 PEDF Secretion(Apical to Basal Ratio) Polarized NA 2.0 2.6 3.1 2.8 2.5 VEGF Secretion(Basal to Apical Ratio)

Example 4

Stability assays were performed on RTA RPE cell therapy compositions.Cells produced according to the methods in Example 1 were suspended inMedia 1 containing 2% DMSO (CS2) or Media 2 containing 5% DMSO (CS5) forup to 3 hours prior to cryopreservation. Therapeutic RPE cells that werecryopreserved after 3 hours incubation in CS2 and CS5 showed similarpost thaw viability, vitality and yield as those cells incubated forless than one hour prior to cryopreservation. The stability results arepresented in Table 2.

TABLE 2 Stability of Therapeutic RPE Cells Post Thaw (Incubation inMedia 1 (CS2) and Media 2 (CS5) Prior to Cryopreservation) IncubationFold Time (hrs) % Vitality Expansion at 2-8° C. % Viability % Yield 24Hrs at Day 14 prior to Post Thaw Post Thaw Post Thaw of Culturepreservation CS2 CS5 CS2 CS5 CS2 CS5 CS2 CS5 0 67 70 73 70  94  92 4.24.8 0.5 67 67 65 72 108 100 4.3 4.5 1 70 66 77 71  92  76 4.8 5.4 2 7067 92 82  89 112 4.9 4.5 3 62 65 64 59  91 112 4.3 4.5

In addition, cells incubated in CS2 or CS5 for 3 hours prior tocryopreservation demonstrated the ability to generate barrier function(tight junctions between RPE cells), measured by the ability to generateTrans Epithelial Electrical Resistance (TEER) above 100Ω and secreteVEGF and PEDF in a polarized manner, as shown in Table 3. (See also FIG.15 ).

TABLE 3 TEER and Polarized Secretion of PEDF and VEGF of TherapeuticRPE® Cells in Media 1 (CS2) and Media 2 (CS5) Post Thaw (Incubation inMedia Prior to Cryopreservation) Polarized Secretion of IncubationBarrier PEDF and VEGF Time Function PEDF Upper VEGF Lower (hrs) at TEER(Ω) to Lower Ratio to Upper Ratio 2-8° C. CS2 CS5 CS2 CS5 CS2 CS5 0 188392 7.1 7.6 1.4 1.8 0.5 211 318 5.5 6.2 1.4 1.9 1 253 347 9.4 9.4 1.51.2 2 107 241 5.1 3.5 1.2 2.7 3 402 715 5.1 5.7 1.7 1.6

Post thawing stability was assessed for RPE cell therapy compositionsdescribed above. RPE cell compositions were formulated in Media 1containing 2% DMSO (CS2) or Media 2 containing 5% DMSO (CS5) andincubated for up to 3 hours prior to cryopreservation. Viability, livecell yield, and potency were determined as described above at timepoints 0 hours, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours and 24 hourspost cryopreservation at between approximately 2 to 8° C.

RPE cells were found to be stable in Media 1 containing 2% DMSO (CS2) orMedia 2 containing 5% DMSO (CS5) for at least about 3 hours prior tocryopreservation and at least about 1 hour post cryopreservation or atleast about 2 hours prior to cryopreservation and at least about 5 hourspost cryopreservation.

Example 5

The safety of three cryopreservation solutions for use with ready toadminister (RTA) RPE cell therapy composition formulations was assessedfollowing sub-retinal injection into Balb/c mice.

A total of 36 Balb/c mice were utilized and divided into four (4) groupsof nine (9) in each group (n=3 for each of the termination time points;1, 3 and 10 days post administration). These groups contained onevehicle (BSS PLUS, Alcon Laboratories) control group and three treatedgroups that received the Test Items (CS5, CS2 and CS2 diluted 1:1 v/vwith BSS PLUS). All animals were administered with the varioustreatments via sub-retinal injection into the left eye. The procedurewas performed under anesthesia using ketamine/Medetomidine at 75 mg/kg,which was given IP two minutes before injection.

During the study, morbidity and mortality, body weight, and generalclinical observation as well external and internal eye examinations wereperformed. Eye evaluation was performed by veterinary ophthalmologistonce during acclimation (baseline measurement prior to dosing) and oneach termination day thereafter. The eye evaluation included:examination of the anterior segment and lens using slit lampbiomicroscopy and examination of the fundus using indirectophthalmoscopy. The animals were sacrificed on days 1, 3 and 10 postdosing and histopathology examination was performed on the injectedeyes.

Clinical, ophthalmologic and histopathological examinations, carried outby a veterinary ophthalmologist and a board of certified veterinarypathologists, demonstrated no major treatment-related or toxicologicallysignificant effects, following sub-retinal administration of RTA RPEformulations in a 10-day follow-up. Histopathological evaluations ofinflammation were based on the presence of neutrophils, lymphocytes,macrophages, and mast cells according to the following criteria: noinflammation as indicated by the absence of inflammatory cells, mildinflammation as indicated by up to 10 cells per ×10 magnification field,moderate inflammation as indicated by between about 10 to 20 cells per×10 magnification field and, strong inflammation as indicated by greaterthan 20 cells per ×10 magnification field.

Histopathological evaluation of the native eye of an untreated animaland the nontreated right eyes of animals in the control groups revealedno pathological changes at all, as shown in FIG. 2 and FIG. 3 .

Histopathological evaluation of the treated eyes (left eyes) revealedthat on termination Day 1, only the animals in the group treated withBSS PLUS:CS2 (1:1 v/v) showed signs of strong inflammation. All otheranimals treated with either BSS PLUS, CS5 or CS2 showed either mild tomoderate inflammation. Images of histopathological slides of the treatedeye taken from an animal treated with BSS Plus and sacrificed on day 1of the study are shown in FIG. 4A and FIG. 4B. These slides show mildinflammation with mild infiltration of the sclera and a few losemacrophages and lymphocytes. (H&E stained at ×4 and ×20 magnificationfield, respectively). FIG. 5A and FIG. 5B show images ofhistopathological slides of the treated eye taken from an animal treatedwith CS5 and sacrificed on day 1. These slides show moderateinflammation with infiltration of the sclera, some macrophages and fewneutrophils. (H&E stained at ×4 and ×20 magnification field,respectively). Images of histopathological slides of the treated eyetaken from an animal treated with CS2 and sacrificed on day 1 of thestudy are shown in FIG. 6A and FIG. 6B. These slides show moderateinflammation with macrophages and neutrophils in the cornea. (H&Estained at ×4 and ×20 magnification field, respectively).

Although most animals treated with either CS2 or BSS PLUS:CS2 displayedminimal fibrin deposition in the anterior chamber at termination Day 1,these acute changes demonstrate a short-term reaction. FIG. 7A shows animage of a histopathological slide of the treated eye taken from ananimal treated with BSS PLUS:CS2 and sacrificed on day 1 of the study,showing strong inflammation with moderate infiltration of the sclera.(H&E stained at ×4 magnification field).

FIG. 7B shows fibrin deposition at the lower right corner next to thelymphocytes in the sclera. (H&E stained at ×20 magnification field).

On termination Day 3, all animals treated with either CS5 or CS2 showeda focal scleral granulomatous reaction (mild to moderate inflammation)characterized by macrophages and dividing fibroblasts. Macrophages werealso observed in animals treated with BSS PLUS, however, they showed adifferent pattern and concentration of cells with no fibroblastactivation and were not related to the injected material. These resultsindicate a typical pattern of early stage reaction to foreign body ingeneral. Consequently, on termination Day 10, all of the animals treatedwith either BSS PLUS, CS5, or CS2 showed no inflammation or mildinflammation and no fibrin deposition. In addition, only one animaltreated with BSS PLUS:CS2 displayed moderate inflammation, while allother animals displayed mild inflammation.

Histopathological images demonstrating moderate inflammation withmoderate infiltration of the sclera and several macrophages in an animaltreated with BSS PLUS and sacrificed on day 3 of the study are shown inFIG. 8A and FIG. 8B. FIG. 9A and FIG. 9B show histopathological imagesof strong inflammation with a focal granulation reaction and severalmacrophages and fibroblasts, which illustrates an early stage,transitory foreign body reaction, in an animal treated with CS5 andsacrificed on day 3 of the study. FIG. 10A and FIG. 10B arehistopathological images from an animal treated with CS2 and sacrificedon day 3 of the study, showing strong inflammation with severalmacrophages and fibroblasts, also demonstrating an early stage,transitory foreign body reaction. Histopathological images from ananimal treated with BSS PLUS:CS2 and sacrificed on day 3 of the study,with mild inflammation and mild edema are shown in FIG. 11A and FIG.11B. FIG. 12A and FIG. 12B show histopathological images from an animaltreated with BSS PLUS and sacrificed on day 10 of the study,illustrating mild inflammation with few macrophages. FIG. 13A and FIG.13B show histopathological images from an animal treated with CS5 andsacrificed on day 10 of the study, illustrating mild inflammation withfew macrophages. FIG. 14A and FIG. 14B show histopathological imagesfrom an animal treated with CS2 and sacrificed on day 10 of the study,illustrating mild inflammation with few macrophages.

The histopathological evaluation results demonstrate that that therewere no major treatment-related and/or toxicologically significanteffects following the sub-retinal administration of BSS PLUS, CS5, CS2,or BSS PLUS:CS2 as compared to the control after 10 days follow-up.Histopathological evaluation of the treated eyes revealed a typicalearly stage reaction to a foreign body on termination Day 3 in groupstreated with BSS Plus, CS5 and CS2. However, this reaction was transientand subsided by Day 10 leaving a very minor macrophage infiltration inthe injected site. Necrosis was not present in any animals' retina orelsewhere.

Example 6

RTA RPE cell therapy compositions were formulated using enzymaticenrichment (isolation/harvest) of pigmented cells and enzymeneutralizing solutions comprising NUTS(−)+Human Serum Albumin (HSA) andNUTS(−) (without Human Serum (HS)) and were analyzed for stabilitybefore and after the addition of cryomedium.

Cells were seeded and expanded in T25, T75 and T175 flasks up to passage4. Upon reaching a polygonality of greater than about 90%, cells wereincubated in TrypLE Select (1X) for up to about 50 minutes at 37° C./5%CO₂. Cells were pooled and placed on ice. Flasks were washed once withequal volume of PBS (−) and the wash was added to the cell pool. The PBS(−) wash was replaced with NUTS (−) for improved enzyme-neutralizing andreduced cell stress.

The cell pool was then sampled (20 μl in 180 μl PBS (−)) and countedusing a cell counter such as the NC-200 cell counter, for example. Thecell pool was then aliquoted into the various quenching solutions forgroups (G1, G2 and G3). Cells from each group were then counted,filtered and aliquoted for cell composition stability analysis at 4° C.

The enzyme neutralizing solution types that were analyzed included:

-   -   Group 1 (G1)— 20% Human Serum (HS)/DMEM (HS positive control        group)    -   Group 2 (G2)— Nutristem (−) with Human Serum Albumin (HSA)    -   Group 3 (G3)— Nutristem (−) (NUTS)

Each of the quenching groups, G1, G2, and G3, were passed through atandem 500-200-40-micron sequential filtering system. Filtered cellsolutions were kept at 4° C. and cell viability was tested at timepoints 0, 2 and 4 hours post-filtration.

At the end of each time point post-filtration, the cells were counted,centrifuged at about 220 g for about 5 minutes, the supernatant wasdiscarded, and the pellet was resuspended in about 5-10 ml CS5, sampledand counted (20 μl in 180 μl 20% HS/DMEM). Based on count results, cellswere diluted in CS5 to obtain a final concentration of 2×10⁶ cells/ml.Cells were placed at 4° C. for different times (0, 2, 3, or 4 hours) forpre-cryopreservation stability analysis, after which the RPEcell+cryomedia compositions were aliquoted into cryovials. Threecryovials were randomly sampled, the cells were counted, andcryopreserved.

Vials were thawed in a 37° C. water bath for about 2.5 minutes. Cellswere immediately sampled for counting (20 μl in 180 μl 20% HS/DMEM) andcell suspensions were diluted by drop-wise addition of warm 20% HS/DMEMculture media. Cells were then washed and placed on ice for additionalanalysis.

Recovery percentages were calculated based on a targeted finalconcentration of 2×10⁶ cells/ml.

Following the sequential filtration, the filtered cell suspensions inthe different enzyme neutralizing solutions, were kept at 4° C. and cellviability at 0, 2 and 4 hours post-filtration was evaluated.

TABLE 4 Cell Composition Viability Post-Filtration Time Avg. % SD Grouppre-centrifuge Viability Viability G1 0 hrs. 98 NA G2 0 hrs. 99 1.2 (n =6) 2 hrs. 99 0.9 4 hrs. 99 0.6 G3 0 hrs. 99 0.5 (n = 6) 2 hrs. 99 0.6 4hrs. 98 1.0

Viability in all groups, at all time points remained at 98% or 99%, asshown in Table 4. No significant differences were found between the twoneutralizing solution formulation groups G2 and G3, at different timepoints (0, 2 and 4 hours) compared to G1 (control group) time point 0hours. FIG. 16 shows the viability of G2 (NUTS(−)+HSA) and G3 (NUTS(−))groups at 4° C. over time post-filtration compared to the control groupG1.

Cell recovery and viability were evaluated within cryopreservationsolution prior to the freezing process (pre-cryopreservation). Celltherapy compositions (post filtration) were kept at 4° C. for timeperiods of 0, 2 and 4 hours. Each solution was then centrifuged andresuspended in cryopreservation solution, CS5, to a final concentrationof 2×10⁶ cells/ml. Next, the cells within the cryopreservation solutions(cryopreservation+cell therapy composition, pre-cryopreservation) werekept at 4° C. for 0, 2, 3 and 4 hours before being aliquoted into 1 mlcryovials. Three vials from each group were sampled and counted toevaluate viability and recovery percentages before vials werecryopreserved.

Cell viability and recovery, before the freezing process, of celltherapy compositions at incubation time 0 hours followed by incubationin cryomedia for 0, 2, 3, and 4 hours are summarized together in Table4A. Tables 4B and 4C include cell compositions incubated for 0, 2, 4hours followed by incubation for 0, 2, 3, 4 hours in cryopreservationsolution (cells+CM). The control group (G1) was evaluated only for timepoint 0 hours for cell compositions and 0 hours for cells+CM,pre-cryopreservation.

TABLE 5A Pre-cryopreservation viability and recovery of cell therapycompositions at 0 hours incubation in neutralizing solution followed byincubation for 0, 2, 3, and 4 hours in cryomedia (Cells + CM). Cells +CM pre-cryo Cells at 0 hours incubation time Group Avg. % Viability SDAvg. % Recovery SD 0 hrs. G1 98 0.9 86 8.1 (n = 3) G2 98 0.8 87 12.5 (n= 6) G3 98 0.9 84 5.3 (n = 6) 2 hrs. G2 98 1.0 86 7.9 (n = 6) G3 97 1.386 12.0 (n = 6) 3 hrs. G2 98 0.6 86 9.5 (n = 6) G3 97 3.4 92 10.4 (n =6) 4 hrs. G2 98 0.9 90 8.7 (n = 6) G3 95 3.3 92 7.8 (n = 6)

As shown in Table 5A, FIG. 17A, and FIG. 17B, at time point 0 hourstherapeutic cell composition incubation followed by 0 hours incubationin cryomedia, pre-cryopreservation, no significant differences inviability and recovery were detected between both neutralizing groups,G2 and G3, compared to the G1 control group. Moreover, 0 hourstherapeutic cell composition incubation in neutralizing solutionfollowed by 2, 3, and 4 hours incubation in cryomedium,pre-cryopreservation, did not present a reduction in viability orrecovery of the cells in both G2 and G3 groups compared to the G1control group. Viability remained above 95% over time and no significantdifferences were observed among the three groups. Furthermore, recoveryof all groups remained in the range of 75%-100%.

TABLE 5B Pre-cryopreservation cell viability and cell recovery after 2hours therapeutic cell composition incubation followed by 0, 2, 3 and 4hours incubation in cryomedium, pre-cryopreservation. Cells + CMpre-cryo Cells at 2 hours incubation time Group Avg. % Viability SD Avg.% Recovery SD 0 hrs. G2 98 0.5 77 2.5 (n = 3) G3 98 0.3 82 8.7 (n = 3) 2hrs. G2 97 1.3 79 3.0 (n = 3) G3 97 1.0 84 3.9 (n = 3) 3 hrs. G2 97 0.983 8.8 (n = 3) G3 98 1.0 76 12.5 (n = 3) 4 hrs. G2 97 2.0 75 9.0 (n = 3)G3 96 0.7 74 4.9 (n = 3)

TABLE 5C Pre-cryopreservation cell viability and cell recovery after 4hours therapeutic cell composition incubation followed by 0, 2, 3, 4hours incubation in cryomedium, pre-cryopreservation. Cells + CMpre-cryo Cells at 4 hours incubation time Group Avg. % Viability SD Avg.% Recovery SD 0 hrs. G2 98 0.4 86 10.4 (n = 3) G3 97 1.2 92 9.5 (n = 3)2 hrs. G2 96 0.2 87 7.9 (n = 3) G3 98 0.2 86 6.4 (n = 3) 3 hrs. G2 982.4 82 8.3 (n = 3) G3 97 0.5 88 7.0 (n = 3) 4 hrs. G2 97 0.9 85 7.8 (n =3) G3 97 1.0 92 8.1 (n = 3)

Tables 5B and 5C and FIG. 18A, FIG. 18B, FIG. 19A, and FIG. 19B showthat when RPE therapeutic cell compositions are incubated inneutralizing solution for 2 and 4 hours followed by 0-4 hours incubationin cryomedium, pre-cryopreservation, similar cell viability and recoveryvalues for both groups G2 (cell compositions in Nutistem(−) with HSA)and G3 (cell compositions in Nutistem(−) (NUTS)) are seen. This assaydemonstrated that there were no significant effects on cell viability orcell recovery given prolonged incubation in either of the neutralizingsolutions or cryomedium, prior to cryopreservation.

Example 7

Cells can experience stress during cryopreservation which may lead topoor survival rates post-thawing. Additionally, stress on the cellsduring harvesting procedures may affect post thawing cell viability andrecovery. Accordingly, cell viability and recovery of RTA therapeuticcell compositions were assessed post-thawing. Therapeutic cellcompositions that were incubated for 0 hours in different neutralizationsolutions (G1, G2, and G3, as described above) followed by incubation incryomedium for 0, 2, 3 and 4 hours pre-cryopreservation were assessedfor viability and recovery post-cryopreservation.

Table 6 summarizes the viability and stability results obtained whentherapeutic cell compositions were incubated in enzyme neutralizingsolutions for 0 hours followed by incubation in cryomedium for 0, 2, 3,and 4 hours (pre-cryopreservation), cryopreserved and then thawed.

TABLE 6 Post thawing viability and recovery of 0 hours cells +neutralizing solution incubation followed by 0, 2, 3, and 4 hoursincubation in cryomedium Pre- Avg. % SD % Avg. % SD % Group SD % GroupSD % Cryopreservation Re- Re- Vi- Vi- % Re- Re- % Vi- Vi- Group time inCS5 covery covery ability ability covery covery ability ability G1 0hrs. 92 7.9 97 1.1 92 7.9 97 1.1 (n = 4) G3 0 hrs. 91 8.8 98 1.0 91 10.097 1.3 (n = 2 hrs. 87 12.6 96 1.3 4-6) 3 hrs. 94 8.0 96 0.9 4 hrs. 958.8 97 1.7 G4 0 hrs. 99 10.1 96 0.8 98 14.7 96 0.7 (n = 2 hrs. 104 18.997 0.4 4-6) 3 hrs. 99 17.1 96 0.9 4 hrs. 89 6.3 96 0.7

Analysis of cell viability and recovery, prior to cryopreservation withdifferent incubation times in cryomedium, revealed that viability wasmaintained above about 90% in all groups at all time points and recoverywas in the range of about 75%-100%.

As shown in FIG. 20A, there were no significant differences in cellviability among the groups and viability remained above about 95% acrossthe entire pre-cryopreservation time range and was comparable to theviability of G1 control group. Analysis of the recovery results, shownin FIG. 20B, revealed higher recovery values for group G4, which peakedat 2 hours pre-cryopreservation and then slightly decreased after 3 and4 hours pre-cryopreservation. However, recovery remained in the range ofabout 80%-100%.

The post thawing results obtained when therapeutic cell compositionswere incubated for 0, 2, and 4 hours in neutralization solution followedby incubation for 0, 2, 3, and 4 hours in cryomedium are presented inTable 7. These results demonstrate the relationship of post-thawingviability and recovery on prolonged exposure of the cells to CS5pre-cryopreservation which, in turn, may be affected by prolongedincubation of the cells in the enzyme neutralizing solution.

TABLE 7 Post thawing viability and recovery of cells incubated in enzymeneutralization solution for 0, 2, 4 hours followed by incubation incryomedia (pre- cryopreservation) for 0, 2, 3, and 4 hours Time DS/DPAvg. % SD % Avg. % SD % Grp % SD % Grp % SD % Group pre-cryo ViabilityViability Recovery Recovery Recov Recov Viab Viab G2 0 hrs/ 95 0.6 770.4 75 3.3 95 1.7 (n = 2) 0 hrs 2 hrs/ 96 1.6 75 1.4 2 hrs 2 hrs/ 96 1.371 7.8 3 hrs 4 hrs/ 93 1.8 79 2.1 4 hrs G3 0 hrs/ 98 1.1 80 5.0 81 7.197 1.3 (n = 2) 0 hrs 2 hrs/ 96 1.4 85 3.5 2 hrs 2 hrs/ 98 1.8 72 13.1 3hrs 4 hrs/ 97 0.1 88 11.0 4 hrs

The viability measured for both groups G2 and G3 was robust at above93%, with no significant differences between groups G2 and G3 across alltime points. Recovery of both groups showed no significant differences.Recovery for group G2 was 75%±3.3 and recovery for group G3 was 81%±7.1.

These results demonstrate that the neutralizing solutions (NUT(−) withHSA (group G2) and without HSA (group G3)) did not compromise cellrecovery or cell viability. No significant differences were foundbetween the two quenching groups with or without HSA (G2 & G3). The cellviability and cell recovery results for the enzyme neutralizingsolutions without HS were comparable to the enzyme neutralizingsolutions comprising HS (G1) both pre- and post-cryopreservation.

In addition, the cell harvesting procedure comprising enzymeneutralizing solutions without HS and a filtration step did notcompromise the viability of the cells (Table 4). Pre-cryopreservationand post thawing analysis of cell recovery and viability showed nosignificant differences between the groups G2 and G3 compared to thecontrol group (G1) (Tables 5-7). The greatest percent recovery andviability were found when cells were incubated in the enzymeneutralizing solutions for up to 2 hours followed by incubation incryomedium for up to 3 hours. However, incubation times of at least 4hours in either the enzyme neutralizing solution or the cryomedium didnot result in a significant decrease in cell viability or cell recovery.

PEDF secretion and cell expansion capability was also measured after thecells were harvested, cryopreserved and thawed. The results are shown inTable 8.

TABLE 8 PEDF secretion and expansion ability of cells post harvestingand cryopreservation for RPE cell compositions with enzyme neutralizingsolution (cells) and RTA RPE cell compositions (cells + CM) incubationtime Time Cells - PEDF Cells + CM LIVE cells Day 14 % Group pre-cryoday-14 ng/ml/day Recovery Assay 1 G1 0-0 3.77E+06 2171 7.54 G2 0-04.07E+06 2452 8.14 0-2 2.80E+06 2407 5.60 0-3 3.21E+06 2255 6.42 0-4 ND2032 ND G3 0-0 ND 2364 ND 0-2 2.73E+06 2181 5.46 0-3 3.48E+06 2739 6.960-4 ND 2310 ND Test Production Run ND 2413 ND Assay 2 G2 0-0 3.54E+062051 7.08 2-2 2.62E+06 2030 5.24 2-3 2.60E+06 2305 5.20 4-4 3.70E+062381 7.40 G3 0-0 3.54E+06 2379 7.08 2-2 2.90E+06 2063 5.80 2-3 3.80E+062289 7.60 4-4 3.15E+06 2382 6.30 Test Production Run 2.35E+06 2404 4.70ND—No Data

As shown in Table 8, upon thawing, cells retained their functionalability to secrete PEDF and to expand. Results indicated no significantdifferences among the two enzyme neutralizing solution groups without HS(G2, G3), at all measured time points or between these groups and theenzyme neutralizing solution group with HS (G1).

Example 8

Therapeutic cell compositions were analyzed for stability (% viabilityand % recovery) post-filtration after incubation at room temperature(RT) and at temperatures between about 2-8° C., as shown in Table 9.

TABLE 9 Incubation times Group 1 Group 2 Group 3 Group 4 Group 5 Cells 2hrs (~4° C.) 2 hrs (~4° C.) 2 hrs (~4° C.) 2 hrs (~4° C.) 2 hrs (RT)Cells + CM T = 0 (~4° C.)  2 hrs (~4° C.) 4 hrs (~4° C.) 6 hrs (~4° C.)6 hrs (RT)

Cells were harvested enzymatically (e.g., TrypLE Select). The enzyme wasneutralized using Nutristem (−) (NUTS). Following sequential filtration,the filtered cell suspension (Cell Pool) was divided into 2 groups, the1^(st) group (“Cells 2-8° C.”) was kept at about 2-8° C. and the 2^(nd)group (“Cells RT”) was kept at RT. The cell viability and cellconcentration of the 2 groups were evaluated at Time 0 and after 2 hoursincubation.

As shown in Table 10, there were no significant differences in cellviability and cell concentration between the groups of cell compositionsincubated pre-cryopreservation at about 2-8° C. or RT. Similarly, therewere no significant differences in percent recovery between the groups,as shown in FIG. 22 .

TABLE 10 Cell viability and cell concentration after 2 hours incubationCell Viability Group concentration (n = 2) Cell Pool 1.30 × 10⁶ 98 Cellsat 2-8° C. 1.26 × 10⁶ 98 Cells at RT 1.25 × 10⁶ 98

After 2 hours incubation, the two groups were centrifuged andresuspended in the cryopreservation solution, CS5, to a finalconcentration of 2×10⁶ cells/ml. The “Cells 2-8° C.” group was dividedinto 4 sub— groups (Cells+CM—groups 1-4), which were incubated at 2-8°C. Cells+CM—group 1 was counted at time point 0 hours for cellconcentration and cell viability and cryopreserved (n=29 cryovials).Cells+CM—group 2 was counted after 2 hours incubation at 2-8° C.,Cells+CM—group 3 was sampled after 4 hours and Cells+CM—group 4 wascounted 6 hours after incubation for cell concentration and cellviability. At the various incubation time points, when counting wascompleted, the Cells+CM—groups 2-4 were cryopreserved by aliquoting 1 mlinto each cryovial (n=30, 30 and 29 cryovials respectively). “Cells+CMRT” was sampled for viability and cell concentration at time points 2,4, and 6 hours. After 6 hours incubation, this group was alsocryopreserved (n=22 cryovials).

TABLE 11 Therapeutic cell composition in cryomedium: cell concentrationand cell viability at various time points. Cell Viability ConditionsGroup concentration (n = 2) % Recovery 4° C. Cells + CM-  2.0 × 10⁶ 97NA group 1 (Time 0) Cells + CM - 2.30 × 10⁶ 95 115 group 2 (2 hrs)Cells + CM - 2.20 × 10⁶ 93 110 group 3 (4 hrs) Cells + CM - 2.30 × 10⁶93 115 group 4 (6 hrs) Room Cells + CM RT  2.0 × 10⁶ 94 NA Temperature(Time 0) (RT) Cells + CM RT 1.70 × 10⁶ 93 85  (2 hrs) Cells + CM RT 1.78× 10⁶ 91 89  (4 hrs) Cells + CM RT 1.73 × 10⁶ 88 86  (6 hrs)

As shown in Table 11 and FIG. 23A and FIG. 23B, there were nosignificant differences in viability between the cellcompositions+cryomedium at RT and cell compositions+cryomedium at about2-8° C. groups, with a small decrease in cell viability in all groupsover time. The results indicate that cell recovery remains stable withinall groups over at least 6 hours in both temperature conditions, about2-8° C. and RT, with a slightly greater recovery of cells incubated atabout 2-8° C., prior to the freezing process. The cells that wereincubated at RT showed a 15% reduction in recovery at 2 hours, however,recovery and viability remained stable for at least 6 hours.

Example 9

Two cell thawing methods, one comprising a water bath at about 37° C.and the other comprising an automated cell thawing unit, were evaluatedbased on cell viability, recovery, sterility, potency and identity, asshown in Table 12.

TABLE 12 Test groups and assay time points for post-thaw analysis.Cells + CM (RTA) Dose Time point (hr.) Group No. (cells/mL) T = 0 T = 2T = 4 Group 1 2 × 10⁶ V, R, I, P V, R, P V, R, P, S Group 2 5 × 10⁶ V,R, P V, R, I, P V, R, P, S Group 3 (Non-GMP) 2 × 10⁶ V, R, P V, R, P V,R, I, P Group 4 (Non-GMP) 5 × 10⁵ V, R, I, P V, R, P V, R, P V—Viability%, R—Recovery %, I—Identity, P—Potency, S—Sterility

Two vials from each of the 4 groups of RTA therapeutic cell compositionswere concurrently thawed; 1 vial using an automated cell thawing unit(e.g., a ThawSTAR automated thawing system by Sigma-Aldrich) and thesecond using a standard water bath at about 37° C. Thawed vials werethen placed on ice. Each vial was gently pipetted and 2 samples of 20 μlfrom each vial were taken, diluted in 180 μl of NUTS (−), vortexed andcounted. Average viability and recovery percentages were calculated foreach thawed vial.

The stability of the RTA therapeutic cell compositions at roomtemperature post-thawing for up to 4 hours was evaluated by assayingthawed cell compositions for viability, recovery, sterility, potency andidentity. Potency was determined by measured TEER, Basal PEDF/VEGF ratiosecretion at day 21 and Apical VEGF/PEDF ratio secretion at day 21.Recovery percentages were calculated based on the targeted finalconcentration of 2×10⁶ or 5×10⁶ cells/ml.

The viability and recovery averages of the RTA therapeutic cellcompositions which were thawed using an automated cell thawing unit werecomparable with those achieved using a conventional water bath at about37° C., at both cell concentrations, as shown in Table 13.

TABLE 13 Average viability and recovery of RTA therapeutic cellcompositions thawed using an automated cell thawing unit vs. thawingusing a water bath at about 37° C. at different cell concentrations.Cell Live cells Avg. Avg. Thawing Concentration concentration Viabil-Recov- Method (cell/mL) (cell/mL) ×10⁶ ity % ery % 37° C. 2 × 10⁶ 2.3295 117 Water (n = 2) Bath 5 × 10⁶ 4.92 95 99 (n = 2) Automated 2 × 10⁶2.29 95 116 Cell (n = 2) Thawing 5 × 10⁶ 5.55 95 112 Unit (n = 2)

As shown in Table 14, the average percent viability for each group attime points 0, 2, and 4 hours was at least 83%. The average percentrecovery of all tested groups was at least about 78% at time points 0,2, and 4 hours. There was a slight decrease of about 4% to 10% inviability and up to a 17% decrease in recovery of all groups post 2 and4 hours room temperature incubation. Average recovery of the 5×10⁶ cellper mL cell concentration was higher than that of the 2×10⁶ cell per mlconcentration at all time points.

TABLE 14 Viability and recovery averages. Cells + Avg. Avg. CM (RTA)Viability % Recovery % Time Point Group No. Vial No. per vial per vial 0Hours Group 1 1 95 104 2 96 99 Group 2 1 96 115 Group 3 1 90 117 Group 41 93 90 2 Hours Group 1 1 87 84 Group 2 1 91 107 Group 3 1 85 117 Group4 1 89 78 4 Hours Group 1 1 85 91 Group 2 1 89 112 Group 3 1 85 107 2 8397 Group 4 1 89 84

FIG. 24A and FIG. 24B are graphs showing the viability and recovery ofthe thawed RTA cell compositions at room temperature over a 4-hourincubation time.

Example 10

The compatibility of the RTA therapeutic cell compositions with adelivery device was assessed. Examples of delivery devises include butare not limited to devices manufactured by the Dutch Ophthalmic ResearchCenter (D.O.R.C) comprising a needle with an outer diameter of about0.63 mm and an inner diameter of about 0.53 mm, a capillary with anouter diameter of about 0.5 mm and an inner diameter of about 0.25 mm,and a tip with an outer diameter of about 0.12 mm and an inner diameterof about 0.07 mm. Delivery device released RTA therapeutic cellcompositions comprising 4 batches were assayed for viability, recoveryand potency following a 2-hour incubation time at RT, post-thawing,using an automated thawing system. All of the RTA cell compositions wereformulated as described in Example 6, except for Group 4, in which nofiltration step prior to cryopreservation was applied. The results arepresented in Table 15.

TABLE 15 Stability of RTA Cell Composition Before Release from DeliveryDevice Identity assay Potency assay % Net TEER PEDF Apical/ VEGF Basal/CRALBP/ (Ω) Basal Ratio Apical Ratio Time point Batch ID PMEL17 (day 14)(day 21) (day 21) 0 Group 1 99.70 605 8.31 2.06 Group 2 NA 525 6.91 2.54Group 3 NA 615 7.08 1.95 Group 4 98.39 322 4.30 2.77 2 hrs. Group 1 NA574 9.71 2.56 Group 2 98.74 536 5.20 2.15 Group 3 NA 660 7.09 2.13 Group4 NA 333 5.21 2.47 4 hrs. Group 1 NA 479 7.62 2.04 Group 2 NA 518 4.102.20 Group 3 98.68 605 6.51 1.99 Group 4 NA 235 3.38 2.34

CRALBP/PMEL17 (identity) values of tested groups (Table 15) were above98% for all tested batches at all time points. Net TEER values (day 14)remained well above 100Ω in all groups. A gradual decrease was apparentas time progressed, mainly in Groups 1, and 4 after 4 hours. Batchesfrom Group 1 and Group 2 were tested for sterility after 4 hoursincubation at Room Temperature and there was no growth detected for bothbatches.

Compatibility of a delivery device to deliver viable, potent RTAtherapeutic cell compositions was evaluated by releasing thawed RTAtherapeutic cell compositions, which were incubated at RT for 2 hours,within the delivery device. First and second dose volumes were tested toallow flexibility. Cells were assayed for viability, recovery, andpotency. Results for the viability and recovery percentages after beingkept at RT for 2 hours before being loading the RTA therapeutic cellcompositions into the delivery device.

TABLE 16 Viability and recovery after 2-hours at time at RT(pre-delivery device). Avg. Viability % Avg. Recovery % Batch ID pervial per vial Group 1 92 91 Group 2 91 94 Group 3 87 94 Group 4 90 76

Post-deliver device release viability and recovery percentage of allgroups are presented in Table 17. Average viability was between 89% and95%. Average total recovery across all groups was between about 71% and94%. There was a slight decrease of up to 8% in recovery of the first100 μl and up to a 16% decrease in the second 100 μl volume comparedwith pre-delivery device recovery results (except in Group 2, whererecovery remained stable).

TABLE 17 Post-delivery device release cell viability and recoveryresult. Avg. Avg. Avg. Viability Recovery Recovery Group Cell % per %per % per ID Conc. Sample sample sample vial Group 1 2 × 10⁶ 100 μl-a 9584 80 100 μl-b 94 75 Group 2 5 × 10⁶ 100 μl-a 91 96 94 100 μl-b 92 93Group 3 2 × 10⁶ 100 μl-a 90 86 84 100 μl-b 89 82 Group 4 5 × 10⁶ 100μl-a 94 73 71 100 μl-b 93 69

Group 4 was formulated with no filtration step prior tocryopreservation. Thus, the reduced recovery may relate to cell andextracellular matrix aggregates residing in the delivery device.

Post-delivery device release net TEER (for day 14) values are shown inTable 18. The net TEER values ranged from between about 154Ω to about435 Ω. The results for the PEDF Apical/Basal Ratio (day 21) and VEGFBasal/Apical Ratio (day 21) are also presented in Table 18.

TABLE 18 Post-delivery device release potency results. PEDF Apical/ VEGFBasal/ Net TEER (Ω) Basal Ratio Apical Ratio Group ID (day 14) (day 21)(day 21) Group 1 331 6.70 1.80 Group 2 159 4.36 1.93 Group 3 435 5.381.90 Group 4 154 2.99 2.19

Although the TEER values for the 2×10⁶ cell dosages for Groups 1 and 3were slightly higher than the TEER values of the 5×10⁶ cell dosages(Groups 2 and 4), all tested post-delivery device sample groupsdisplayed biological activity, according to potency assay results.

Example 11

Cryoshippers were loaded with liquid nitrogen, prepared for transportand loaded with the batches of cryopreserved RTA therapeutic cellcompositions described in Example 10 (Groups 1-4). All of the RTA cellcompositions were formulated as described in Example 6, except for Group4, in which no filtration step prior to cryopreservation was applied.RTA therapeutic cell compositions were shipped from a Jerusalem, Israelto a US-biorepository in Frederick, Md., with intermediate storage inthe vapor phase of a liquid nitrogen freezer and shipment of the productback to Jerusalem, Israel. The shipments included air and groundtransportation of 4 batches (Groups 1-4) stored in a single vapor-phasecryoshipper and was carried out by World Courier. The Cryoshipperprovided the required storage conditions of approximately (−196° C.) to(−150° C.) and was monitored by an internal Data Logger. Groups 1-4 wereassayed for appearance, viability, recovery, potency and sterility. Theresults are presented in Table 19.

TABLE 19 Appearance, viability, recovery, potency and sterility ofcryoshipped RTA cell compositions. Assay Group 1 Group 2 Group 3 (2 ×10⁶ cells/ml) (5 × 10⁶ cells/ml) (2 × 10⁶ cells/ml) Homogenous, opaquecell Homogenous, opaque cell Homogenous, opaque cell Group 4 suspension,free of visible suspension, free of visible suspension, free of visible(5 × 10⁶ cells/ml) foreign particles and foreign particles and foreignparticles and * Clumps Appearance non-dissociated aggregatesnon-dissociated aggregates non-dissociated aggregates were observed %Viability (Avg.) 95%  94%  91% 90% % Recovery (Avg.) 98% 110% 108% 75%TEER (Ω) (day 14) 672 522 658 395 PEDF Apical/Basal 7.73 8.14 7.59 4.89Ratio (day 21) VEGF Basal/Apical 1.97 2.35 2.26 2.44 Ratio (day 21) 14days Sterility No growth No growth Not tested Not tested * The clumpswere the result of formulation without filtration before fill and finishprocedure.

Although the temperature in the cryoshipper did reach to below −196° C.,this temperature does not pose a risk to the integrity and quality ofthe cell composition and is within the calibrated range of liquidnitrogen data loggers.

The results indicate that the stability, quality and integrity ofcryopreserved RTA cell compositions formulated in CS5 cryomedium aremaintained during controlled shipping from a Jerusalem, Israel to aUS-biorepository in Frederick, Md., with intermediate storage in thevapor-phase of a liquid nitrogen freezer and shipment of the productback to Jerusalem, Israel.

Example 12

The safety of RTA therapeutic cell compositions comprising RPE cells andcryopreservation solution was assessed following sub-retinal injectioninto NOD/SCID mice. A total of 40 female NOD/SCID mice at the age of 5-9weeks were utilized and divided into four (4) groups of 4 or 12 animalsin each group (n=1 or 3 for each of the termination time points; 1, 3,7, and 14 days post administration).

Four compositions were evaluated. Group 1 was administered BSS Plus,Group 2 was administered CS5 cryomedium, Group 3 was administered RTAtherapeutic cell composition (comprising RPE cell and CS5), and Group 4was administered RPE cells in BSS Plus, as shown in Table 20.

TABLE 20 Experimental design. Dose Group No. of Volume Cell Admin- No.Animals Treatment (μl/mouse) Concentration istration 1 4 BSS Plus 1 μlN/A Subretinal 2 12 CS5 1 μl N/A 3 12 RTA 1 μl 5 × 10³ (Cells +cells/mouse CS5) 4 12 BSS Plus + 1 μl 5 × 10³ Cells cells/mouse

Animals were observed periodically during the first 24 hours (withspecial attention given during the first 4 hours post dosing), and dailythereafter, until termination. Observations performed for any changes inlocal injection site, skin, fur, eyes, mucous membranes, respiratory,occurrence of secretions and excretions (e.g. diarrhea) and autonomicactivity (e.g. lacrimation, salivation, piloerection, pupil size,unusual respiratory pattern). Changes in gait, posture and response tohandling, as the presence of bizarre behavior, tremors, convulsions,sleep and coma are also included. No observed abnormalities, toxicsigns, moribund condition and unscheduled deaths were recorded. Eyeexamination was performed by veterinary ophthalmologist once duringacclimation, and on each termination day. In all right eyes(non-treated) from all groups, no visible lesions (NVL) were observed.

At termination, animals were sacrificed by CO2 asphyxiation and grosspathology was performed examining the local injection site (eyes)including the different eye structures, major tissue and organ systems.Animals were enucleated, including the optic nerve, and fixed inDavidson's solution. All eyes were subject to histopathologicexaminations.

Histopathologic evaluation revealed the presence of macrophageinfiltration in a few cell-treated animals from Groups 3 and 4, on Days3 and 14 (1 of 3 animals in each termination day and group). Thesefindings are probably due to a late immune response to the injection ofhuman RPE cells. However, most animals in the cell-treated groups(20/24) did not show any immune response to the transplanted cells. InGroups 1 and 2, which were not treated with cells, neutrophils wereobserved on Days 1, 3 and 7 (in 3 animals). Neutrophils are common inearly stages of the immune response, while the lack of macrophagesindicate no late response. Except for the appearance of macrophages in afew of the cells-treated mice (4/24), all groups displayed similarpathologies, indicating that the findings were most likely related to aprocedure-induced inflammation. FIG. 25A and FIG. 25B are representativehistological images at ×4 and ×20 magnification, respectively, showingmild inflammation in an animal treated with RTA formulation (cells+CS5).Based on the collected data, there were no major treatment-relatedand/or toxicologically significant effects following the sub-retinaladministration of the therapeutic cell compositions as compared to thevehicle after 10 days follow-up.

Example 13

To evaluate the comparability of RTA RPE therapeutic cell compositionsformulated during different manufacturing runs, several batches wereprepared. hESCs were mechanically passaged by first thawing at least oneampule of hESCs. Post thawing, 10 fragments retrieved from the ampule,were seeded on two Center Well (CW) plates containing a monolayer ofirradiated human cord derived feeder cells and incubated in ‘NutriStemPlus’+HSA medium (or equivalent) at 37° C./5% CO₂ (1 ampule→2 CW with 10colony fragments).

hESC colonies were expanded and passaged once a week for about 3 moreweeks until reaching a total of 45 center well plates. Colonies werethen transferred to 6 cm plates with feeders at a ratio of about 2:1 (2CW→6 cm plate) and cultured for about 6 days in ‘NutriStem Plus’+HSAmedium at 37° C./5% CO₂.

To form spheroid bodies (SBs) and start the RPE directed differentiationprocess, hESC colonies were collected from the 6 cm plates andtransferred to plates (such as Hydrocell™ (Nunc)) (non-adherent surface)at a ratio of about 5:1 (5×6 cm plate 4 μlate) and cultured for about 1week in ‘NutriStem Minus’ medium supplemented with Nicotinamide at 37°C./5% CO₂/5% O₂. About one week after SB formation, SBs were collectedand broken down to smaller fragments by pipetting. SB fragments wereseeded on 6 well plates coated with Laminin 511 (BioLamina, Stockholm,Sweden) or an equivalent and cultured in the presence of Nicotinamideand Activin A (combination of Nicotinamide and Activin A variedaccording to the differentiation stage) for about 5 weeks in NuriStemMinus or equivalent at about 37° C./5% CO₂/5% O₂ and about 1 week at 37°C./5% CO₂. At the end of the differentiation stage, pigmented cells wereenriched enzymatically and transferred to recombinant human Gelatincoated T175 (175 cm²) flasks (P0) and incubated in NutriStem Minus (20%human serum/DMEM for the first 2-3 days) at 37° C./5% CO₂.

Cells may be harvested around day 14 and passaged to rh-Gelatin coatedT175 flasks and incubated in NutriStem Minus (20% human serum/DMEM forthe first 2-3 days) at about 37° C./5% CO₂ (P1). Cells were harvested onday 12 and passaged to rh-Gelatin coated T175 flasks and incubated inNutriStem Minus (20% human serum/DMEM for the first 2-3 days) at 37°C./5% CO₂ (P2).

On day 10 post P2 passage, the T175 flasks were harvested, filteredusing sequential tandem of 500-200-40 μm cell strainer system and thenpooled. The total number of cells at this point can be at least about551×10⁶ cells. Cells were centrifuged and resuspended in cryomedium(such as CryoStor 5 (BioLife Solutions Inc., Bothell, Wash.) forexample) and counted to reach a final concentration of about 1×10⁶cell/ml, about 2×10⁶ cell/ml, about 3×10⁶ cell/ml, about 4×10⁶ cell/ml,about 5×10⁶ cell/ml, about 6×10⁶ cell/ml, about 7×10⁶ cell/ml, about8×10⁶ cell/ml, or about 9×10⁶ cell/ml. This cell suspension can be keptat 2-8° C. until it is dispensed into cryovials. The RTA therapeuticcell composition can then by cryopreserved using a controlled-ratefreezer and then transferred to vapor-phase LN2 freezer. Examples offreezing profiles that may be used include:

-   -   Wait at 4° C.    -   Hold for 1 minute at 4° C.    -   1.00° C./minute Sample to −11° C.    -   30.00° C./minute Chamber to −50° C.    -   15.00° C./minute Chamber to −25° C.    -   1.00° C./minute Chamber to −50° C.    -   10.00° C./minute Chamber to −90° C.    -   End        or    -   Wait at 4° C.    -   1.00° C./minute Sample to −4° C.    -   25.00° C./minute Chamber to −40° C.    -   10.00° C./minute Chamber to −12° C.    -   1.00° C./minute Chamber to −40° C.    -   10.00° C./minute Chamber to −90° C.    -   End

Table 21 provides morphology and purity results of the hESCs as they areexpanded, differentiated into RPE cells and expanded as RPE cells. Asshown in Table 21, the hESCs were successfully expanded anddifferentiated using the process described above.

TABLE 21 Analysis of cells during the expansion and differentiationphases for RTA Batch A Step Test Result End of hESCs Morphologyassessment   70% expansion Pluripotent Markers analysis 66.16%(TRA-1-60+/OCT4+) End of Purity (CRALBP+/PMEL17+) 26.77%differentiation/ Enzymatic Isolation End of P0 (RPE Purity(CRALBP+/PMEL17+) 88.95% expansion start) End of P1 (RPE Purity(CRALBP+/PMEL17+) 98.85% expansion middle) hESC impurities (TRA-1-0.00000% BLOD 60+/OCT4+) End of P2 (RPE Morphology assessment Confluentand expansion end) polygonal Purity (CRALBP+/PMEL17+) 98.37%

In addition, percent viability, cell concentration, percent recovery,and purity were determined for Batch A. Results are presented in Table22.

TABLE 22 Percent viability, cell concentration, percent recovery, andpurity for RTA Batch A Test Result Viability ± S.E  95% ± 0.97% Totalcells/1 mL ± S.E 2.36 × 10⁶ ± 5.86 × 10⁴ % Recovery ± S.E 118% ± 2.93%Purity (CRALBP+/PMEL17+) 98.74% hESC impurities (TRA-1- 0.00000% BLOD60+/OCT4+)

Additional batches were produced and analyzed as described for Batch A.The cell dose for each Batch is shown in Table 23.

TABLE 23 Cell doses for each Batch Batch ID Cell Dose Batch B 5 × 10⁶cells/ml Batch C 5 × 10⁶ cells/ml Batch D 2 × 10⁶ cells/ml Batch E 2 ×10⁶ cells/ml Batch F 5 × 10⁶ cells/ml

Results for Batches B-F are presented in Table 24 and Table 25.

TABLE 24 Morphology, sterility, and purity for Batches B-F Step TestResult End of Morphology assessment   70% hESCs Sterility No Growthexpansion Mycoplasma No Growth Endotoxin <3.84 EU/ml  PluripotentMarkers Analysis 78.30% (TRA-1-60+/OCT4+) End of P1 Purity(CRALBP+/PMEL17+) 99.76% (middle of hESC impurities (TRA-1- Not DetectedRPE expansion) 60+/OCT4+) End of P2 Morphology assessment Confluent and(end of RPE polygonal expansion) Purity (CRALBP+/PMEL17+) 99.81%Sterility* Batch B No Growth Batch C No Growth Batch D No Growth Batch ENo Growth Batch F No Growth Mycoplasma* Batch B No Growth Batch C NoGrowth Batch D No Growth Batch E No Growth Batch F No Growth EndotoxinBatch B 0.03 EU/ml (LAL)* Batch C 0.03 EU/ml Batch D 0.03 EU/ml Batch E0.03 EU/ml Batch F 0.03 EU/ml *Cell composition tested

TABLE 25 Percent viability, cell concentration, percent recovery,purity, sterility, PEDF secretion and VEGF secretion for Batches B-FBatch ID Test Result Batch B Percent Viability ± SE   95% ± 0.46% BatchC   95% ± 0.37% Batch D   96% ± 1.15% Batch E   97% ± 0.22% Batch F  96% ± 0.25% Batch B Total cells/1 mL ± SE 5.56 × 10⁶ ± 1.38 × 10⁵Batch C 5.37 × 10⁶ ± 2.53 × 10⁵ Batch D 1.89 × 10⁶ ± 1.08 × 10⁵ Batch E2.25 × 10⁶ ± 8.74 × 10⁴ Batch F 5.81 × 10⁶ ± 6.90 × 10⁴ Batch B PercentRecovery ± SE 107% ± 2.23% Batch C 102% ± 5.17% Batch D  91% ± 5.92%Batch E 109% ± 3.89% Batch F 112% ± 1.44% Batch B Purity(CRALBP+/PMEL17+) 99.10% Batch C 99.36% Batch D 99.39% Batch E 99.52%Batch F 99.40% Batch B hESC impurities (TRA-1-60+/ Not Detected OCT4+)Batch B Sterility No Growth Batch C No Growth Batch D No Growth Batch ENo Growth Batch F No Growth Batch B PEDF secretion (ng/ml/day)-Day 7477.77 VEGF secretion (ng/ml/day)-Day 7 1.59 PEDF secretion(ng/ml/day)-Day 14 2458 VEGF secretion (ng/ml/day)-Day 14 5.90 Batch CPEDF secretion (ng/ml/day)-Day 7 688.18 VEGF secretion (ng/ml/day)-Day 71.97 PEDF secretion (ng/ml/day)-Day 14 2040 VEGF secretion(ng/ml/day)-Day 14 5.14 Batch D PEDF secretion (ng/ml/day)-Day 7 614.22VEGF secretion (ng/ml/day)-Day 7 1.61 PEDF secretion (ng/ml/day)-Day 142468 VEGF secretion (ng/ml/day)-Day 14 5.68 Batch E PEDF secretion(ng/ml/day)-Day 7 481.75 VEGF secretion (ng/ml/day)-Day 7 1.28 PEDFsecretion (ng/ml/day)-Day 14 2117 VEGF secretion (ng/ml/day)-Day 14 5.20Batch F PEDF secretion (ng/ml/day)-Day 7 200.21 VEGF secretion(ng/ml/day)-Day 7 0.79 PEDF secretion (ng/ml/day)-Day 14 2016 VEGFsecretion (ng/ml/day)-Day 14 4.44

As shown in Table 24 and Table 25, the formulation method of RTA RPEtherapeutic cell compositions is reproducible and robust with regard tocell morphology, sterility, purity, percent viability, cellconcentration, percent recovery, PEDF secretion and VEGF secretion(potency).

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

1-79. (canceled)
 80. A composition for treatment of a retinal conditioncomprising: (a) a purine nucleoside, a branched glucan, a zwitterionicorganic chemical buffering agent, and dimethyl sulfoxide (DMSO); and (b)retinal pigment epithelial (RPE) cells, wherein the composition isformulated to be stored at cryothermic temperatures and wherein thecomposition is ready to administer to a subject directly after thawing.81. A method of treating a retinal condition in a subject in needthereof, the method comprising administering to the subject acomposition comprising: (i) a purine nucleoside, a branched glucan, azwitterionic organic chemical buffering agent, and dimethyl sulfoxide(DMSO); and (ii) retinal pigment epithelial (RPE) cells, wherein thecomposition can be stored at cryothermic temperatures and wherein thecomposition is ready to administer to a subject directly after thawing.82. A method of formulating an RPE cell composition, comprising: a)suspending RPE cells to form a cell suspension in a cell preservationmedia comprising a purine nucleoside, a branched glucan, a zwitterionicorganic chemical buffering agent, and dimethyl sulfoxide (DMSO); and b)storing the cell suspension at a cryothermic temperature; wherein atleast about 60% of the cells are viable after thawing the cellsuspension.
 83. The method of claim 82, wherein the RPE cell compositionis ready to administer to a subject directly after thawing.
 84. Themethod of claim 82, wherein the purine nucleoside is adenosine, thebranched glucan is dextran-40, the zwitterionic organic chemicalbuffering agent is HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), and the cell preservation mediafurther comprises L-glutathione.
 85. The method of claim 82, wherein theRPE cell composition is for the treatment of a retinal condition. 86.The method of claim 85, wherein the retinal condition comprisesnon-exudative age related macular degeneration (AMD).
 87. The method ofclaim 82, further comprising loading the RPE cell composition into adelivery device after thawing.
 88. The method of claim 87, wherein thedelivery device comprises a needle.
 89. The method of claim 82, the RPEcell composition comprising between about 0.5% and about 7% DMSO. 90.The method of claim 82, the RPE cell composition comprising betweenabout 1.5% and about 6.5% DMSO.
 91. The method of claim 82, the RPE cellcomposition comprising between about 1.5% and about 3% DMSO.
 92. Themethod of claim 82, the RPE cell composition comprising between about 4%and about 6% DMSO.
 93. The method of claim 82, wherein the RPE cellcomposition is not washed or reconstituted prior to administering to thesubject.
 94. The method of claim 82, wherein dead cells are not removedfrom the RPE cell composition prior to administration to the subject.95. The method of claim 82, wherein the RPE cell composition is storedat the cryothermic temperatures from about 1 month to about 48 months.96. The method of claim 82, wherein the cryothermic temperature is fromabout −4° C. to about −200° C., or from about −20° C. to −200° C., orfrom about −70° C. to −196° C., or from about −4° C. to −196° C.
 97. Themethod of claim 82, wherein the RPE cells demonstrate one or more oftight junctions, generation of blood-retinal barriers, or polarized PEDFand VEGF secretion after administration to the subject.
 98. The methodof claim 82, wherein the RPE cell composition further comprises one ormore of: a sugar acid, a base, an antioxidant, a halide salt, a basicsalt, a phosphate salt, a sugar, a sugar alcohol, and water.
 99. Themethod of claim 98, wherein the sugar acid comprises lactobionic acid,glyceric acid, xylonic acid, gluconic acid, ascorbic acid, neuraminicacid, ketodeoxyoctulosonic acid, glucuronic acid, galacturonic acid,galacturonic acid, iduronic acid, tartaric acid, mucic acid, orsaccharic acid.
 100. The method of claim 98, wherein the base comprisessodium hydroxide, or potassium hydroxide.
 101. The method of claim 98,wherein the antioxidant comprises L-glutathione, ascorbic acid, lipoicacid, uric acid, a carotene, alpha-tocopherol, or ubiquinol.
 102. Themethod of claim 98, wherein the halide salt comprises potassiumchloride, sodium chloride, or magnesium chloride.
 103. The method ofclaim 98, wherein the basic salt comprises potassium bicarbonate, sodiumbicarbonate, or sodium acetate.
 104. The method of claim 98, wherein thephosphate salt comprises potassium phosphate, sodium phosphate, orpotassium phosphate.
 105. The method of claim 98, wherein the one ormore sugars comprises dextrose, sucrose.
 106. The method of claim 98,wherein the sugar alcohol comprises mannitol, sorbitol, erythritol orxylitol.
 107. The method of claim 82, wherein the RPE cell compositioncomprises: adenosine, dextran-40, lactobionic acid, HEPES(N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)), sodiumhydroxide, L-glutathione, potassium chloride, potassium bicarbonate,potassium phosphate, dextrose, sucrose, mannitol, calcium chloride,magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), and water.
 108. The method of claim 82, wherein atleast about 60% to about 75%, or at least about 60% to about 92%, or atleast about 60% to about 95%, or at least about 62% to about 70% of thecells are viable after thawing, alternatively, wherein at least about70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% of the cells are viable after thawing.